'••. 


BIOLvOGY 

LIBRA  iv  i 

G 


MANUAL 


OF 


BACTERIOLOGY 


ROBERT  MUIR,  M.A.,  M.D.,  F.R.C.R(ED.) 

I'KOFKSSOR  OK  PATHOLOGY,    UNIVERSITY  OF  GLASGOW 


AM» 


JAMES  RITCHIE,  M.A.,  M.D.,  F.R.C.P.(Ea) 

MI'KltlNTKNDK.XT  OK   TI1K   ROYAL  COLLKGK  OK    PHYSICIAN'S*   LABORATORY,   KDIXBrROII 
H.UMKIM.V    IMiolKSSOR    OK    1'ATHOLOOY    IX    TIIK    I'XIVKRSITY    OK   OXKORIt 


FIFTH  EDITION 


WITH  ONE  HUNDRED  ,(•  SKVEXTY-FOUR  IL7ArSTJiATIONS 

7AT  Till-   TKXT 
AND  SIX  COLOURED  PL  AT  EX 


NEW  YORK 
THE  MACMILLAN  COMPANY 


Main  Lib,  I   Of  /  r 

Agric.  Dept. 

fi'OLOGY 

LIBRARY 


PREFACE  TO  THE  FIFTH  EDITION. 


DURING  the  three  years  which  have  elapsed  since  the  publication 
of  the  previous  edition,  important  additions  to  knowledge  have 
been  made  in  nearly  every  department  of  bacteriological  research. 
We  have  again  endeavoured  to  incorporate  these  as  fully  as 
possible,  having  in  view  our  primary  object,  namely,  to  supply  a 
Manual  for  students  and  practitioners  of  medicine. 

It  is  impossible  to  refer  in  detail  to  the  new  matter  introduced, 
as  this  occurs  in  practically  every  chapter.  It  may,  however,  be 
mentioned  that  in  dealing  with  technique,  we  have  introduced  a 
new  chapter  in  which  the  more  recent  methods  of  investigating 
the  properties  of  serum  and  allied  subjects  are  described.  While 
it  is  necessary  for  students  of  medicine  to  have  a  general  know- 
ledge of  such  subjects,  those  commencing  independent  bacterio- 
logical research  may  benefit  from  the  details  given  in  this 
department. 

\Vi-  have  also  in  the  present  edition  grouped  together  in  a 
new  chapter  the  pathological  conditions  with  which  spirochyetes 
are  associated,  but  as  the  biological  relationships  of  these  organ- 
isms to  kindred  forms  are  still  not  completely  determined,  we 
have  retained  this  chapter  in  its  former  position. 

We  have  transferred  the  consideration  of  Yellow  Fever  to  the 
Appendices,  which  include  diseases  of  protozoal  origin  and  con- 
•  lit  ions  in  which  the  nature  of  the  infective  agent  is  still  un- 
known. In  further  following  this  principle,  we  have  added 

258'809 


vi  PREFACE  TO  THE  FIFTH  EDITION 

new  Appendices  dealing  with  Acute  Poliomyelitis,  Phlebotomus 
Fever,  and  Typhus  Fever. 

Several  new  illustrations  will  be  found  in  the  text,  and  we 
have  also  added  a  series  of  coloured  plates  which  have  been 
reproduced  from  drawings  by  Mr.  Richard  Muir  of  the  Depart- 
ment of  Pathology,  University  of  Edinburgh.  f 

November  1910. 


PREFACE  TO  THE  FIRST  EDITION. 


THE  science  of  Bacteriology  has,  within  recent  years,  become 
so  extensive,  that  in  treating  the  subject  in  a  book  of  this  size 
we  are  necessarily  restricted  to  some  special  departments,  unless 
the  description  is  to  be  of  a  superficial  character.  Accordingly, 
as  this  work  is  intended  primarily  for  students  and  practitioners 
of  medicine,  only  those  bacteria  which  are  associated  with 
disease  in  the  human  subject  have  been  considered.  We  have 
made  it  a  chief  endeavour  to  render  the  work  of  practical  utility 
for  beginners,  and,  in  the  account  of  the  more  important 
methods,  have  given  elementary  details  which  our  experience  in 
the  practical  teaching  of  the  subject  has  shown  to  be  necessary. 

In  the  systematic  description  of  the  various  bacteria,  an 
attempt  has  been  made  to  bring  into  prominence  the  evidence 
of  their  having  an  etiological  relationship  to  the  corresponding 
diseases,  to  point  out  the  general  laws  governing  their  action  as 
producers  of  disease,  and  to  consider  the  effects  in  particular 
instances  of  various  modifying  circumstances.  Much  research 
on  certain  subjects  is  so  recent  that  conclusions  on  many  points 
must  necessarily  be  of  a  tentative  character.  We  have,  therefore, 
in  our  statement  of  results  aimed  at  drawing  a  distinction 
between  what  is  proved  and  what  is  only  probable. 

In  an  Aj-pemlix  we  have  treated  of  four  diseases;  in  two  of 
tin -so  the  causal  organism  is  not  a  bacterium,  whilst  in  the  other 
two  its  nature  is  not  yet  determined.  These  diseases  have  been 

vii 


viii  PREFACE  TO  THE  FIRST  EDITION 

included  on  account  of  their  own  importance  and  that  of  the 
pathological  processes  which  they  illustrate. 

Our  best  thanks  are  due  to  Professor  Greenfield  for  his  kind 
advice  in  connection  with  certain  parts  of  the  work.  We  have 
also  great  pleasure  in  acknowledging  our  indebtedness  to 
Dr.  Patrick  Manson,  who  kindly  lent  us  the  negatives  or  pre- 
parations from  which  Figs.  163-168  have  been  executed. 

As  we  are  convinced  that  to  any  one  engaged  in  practical 
study,  photographs  and  photomicrographs  supply  the  most  useful 
and  exact  information,  we  have  used  these  almost  exclusively  in 
illustration  of  the  systematic  description.  These  have  been 
executed  in  the  Pathological  Laboratory  of  the  University  of 
Edinburgh  by  Mr.  Richard  Muir.  The  line  drawings  were 
prepared  for  us  by  Mr.  Alfred  Robinson,  of  the  University 
Museum,  Oxford. 

To  the  volume  is  appended  a  short  Bibliography,  which, 
while  having  no  pretension  to  completeness,  will,  we  hope/  be  of 
use  in  putting  those  who  desire  further  information  on  the  track 
of  the  principal  papers  which  have  been  published  on  each  of 
the  subjects  considered. 

June  1897. 


CONTENTS. 

CHAPTER   I. 
GENERAL  MORPHOLOGY  AND  BIOLOGY. 

PAGE 

INTRODUCTORY — Terminology — Structure  of  the  bacterial  cell — 
Reproduction  of  bacteria  —  Spore  formation  —  Motility  — 
Minuter  structure  of  the  bacterial  protoplasm  —  Chemical 
composition  of  bacteria  —  Classification  —  Food  supply — Re- 
lation of  bacteria  to  moisture,  gaseous  environment,  tempera- 
ture, and  light  —  Conditions  affecting  bacterial  motility  — 
Effects  of  bacteria  in  nature— Methods  of  bacterial  action- 
Variability  among  bacteria  .....  1 

CHAPTER  II. 
METHODS  OP  CULTIVATION  OF  BACTERIA. 

Introductory — Methods  of  sterilisation  —  Preparation  of  culture 
media — Use  of  the  culture  media — Methods  of  the  separation 
of  aerobic  organisms— Principles  of  the  culture  of  anaerobic- 
organisms  —  Miscellaneous  methods  —  General  laboratory 
rules  ........  26 

CHAPTER  III. 
MICROSCOPIC  METHODS. 

The  microscope — Examination  of  hanging-drop  cultures — Film  pre- 
parations— Examination  of  bacteria  in  tissues — The  cutting 
of  sections— Staining  principles— Mordants  and  decolorisers 
— Formula?  of  stains— Gram's  method  and  its  modifications 
— Stain  for  tubercle  and  other  acid-fast  bacilli— Staining  of 
spores,  capsules,  and  flagella— The  Komaiiowsky  stains  .  Ul 


CONTENTS 


CHAPTER  IV. 

EXAMINATION  OF  SERUM — PREPARATION  OF  VACCINES — 
GENERAL  BACTERIOLOGICAL  DIAGNOSIS— INOCULATION 
OF  ANIMALS. 

PAGK 

Observation  of  agglutination  and  sedimentation — Opsonic  methods 
— Method  of  measuring  the  phagoeytic  capacity  of  the  leuco- 
cytes— Bactericidal  methods — Hfemolytic  tests — Fixation  and 
deviation  of  complement — Wassermann  reaction — Preparation 
of  vaccines — Wright's  method  of  counting  bacteria  in  dead  cul- 
tures— General  bacteriological  diagnosis — Routine  procedure 
— Inoculation  of  animals — Autopsies  on  animals  .  .  117 


CHAPTER  V. 
BACTERIA  IN  AIR,  SOIL,  AND  WATER.    ANTISEPTICS. 

Air  :  Methods  of  examination — Soil :  Methods  of  examination — 
Varieties  of  bacteria  in  soil.  Water  :  Methods  of  examination 
— Bacteria  in  water — Bacteriology  of  sewage — Antiseptics  : 
Methods  of  investigation — The  action  of  antiseptics — Certain 
particular  antiseptics  .  .  .  .  .  .147 

CHAPTER  VI. 

RELATIONS  OF  BACTERIA  TO  DISEASE — THE  PRODUCTION 
OF  TOXINS  BY  BACTERIA. 

Introductory  —  Conditions  modifying  pathogenicity  —  Modes  of 
bacterial  action — Tissue  changes  produced  by  bacteria — Local 
lesions  —  General  lesions  —  Disturbances  of  metabolism  by 
bacterial  action — The  production  of  toxins  by  bacteria,  and 
the  nature  of  these — Allied  vegetable  and  animal  poisons — 
The  theory  of  toxic  action  .....  175 

CHAPTER  VII. 
INFLAMMATORY  AND  SUPPURATIVE  CONDITIONS. 

The  relations  of  inflammation  and  suppuration — The  bacteria  of 
inflammation  and  suppuration— Experimental  inoculation — 
Lesions  in  the  human  subject — Mode  of  entrance  and  spread 
of  pyogeuic  bacteria — -Ulcerative  endocarditis — Acute  suppur- 


CONTENTS  xi 


ative  periostitis—  Erysipelas  —  Conjunctivitis  —  Acute  rheu- 
matism— Vaccination  treatment  of  infections  by  the  pyogenic 
cocci — Methods  of  examination  in  inflammatory  and  suppur- 
ative  conditions ...  .  200 


CHAPTER  VIII. 
INFLAMMATORY  AND  SUPPURATIVE  CONDITIONS,  continued : 

THE  ACUTE  PNEUMONIAS,  EPIDEMIC  CEREBRO-SPINAL 
MENINGITIS. 

Introductory  —  Historical  —  Bacteria  in  pneumonia  —  Fraenkel's 
pncumococcus — Fried laender's  pneumococcus — Distribution  of 
pnrumohacteria —  Experimental  inoculation  —  Pathology  of 
pneumococcus — Methods  of  examination.  Epidemic  cerebro- 
spinal  meningitis— Serum  reactions— Allied  diplococci  .  224 

CHAPTER     X. 

GONORRHOEA  AND  SOFT  SORI:. 

The  gonococcus  —  Microscopical  characters  —  Cultivation  —  Com- 
parison with  meningococcus  —  Relations  to  the  disease  —  Its 
toxin — Distribution — Gonococcus  in  joint  affections — Methods 
of  diagnosis — Soft  sore — Microscopical  characters  and  culti- 
vation of  bacillus  ......  249 

CHAPTER    X. 
TUBERCULOSIS. 

IlMnrical — Tuberculosis  in  animals — Tubercle  bacillus — Staining 
relictions— Cultivation  of  tubercle  bacillus— Powers  of  resist- 
ance—Action  on  the  tissues — Histology  of  tuberculous  nodules 
— Distribution  of  bacilli — Bacilli  in  tuberculous  discharges — 
Experimental  inoculation  —  Varieties  of  tuberculosis  —  Other 
acid-fast  bacilli — Action  of  dead  tubercle  bacilli — Sources  of 
human  tuberculosis— Specific  reactions  of  the  tubercle  bacillus 
—Phenomena  of  supersensitiveness — Tuberculin  reactions — 
Toxins  of  the  tubercle  bacillus — Koch's  tuberculin — Immunity 
phenomena  in  tuberculosis— Koch's  Tuberculin-R — Therapeutic 
application  of  the  tuberculins — Active  inimuni*ation  associated 
with  opsonic  observations — Antitubercular  sera — Methods  of 
••xaiiiiiialiuii  260 


xii  CONTENTS 

CHAPTER  XL 
LEPROSY. 

PAGE 

Pathological  changes — Bacillus  of  leprosy — Position  of  the  bacilli 

— Relations  to  the  disease — Methods  of  diagnosis         .  .       297 

CHAPTER  XII. 

GLANDERS  AND  RHINOSCLEROMA. 

Glanders  :  The  natural  disease — The  glanders  bacillus — Cultiva- 
tion of  glanders  bacillus — Powers  of  resistance — Experimental 
inoculation — Action  on  the  tissues — Mode  of  spread — Serum 
reactions — Mallein  and  its  preparation — Methods  of  examina- 
tion— Rhinoscleroma  .  .  .  .  .  306 


CHAPTER  XIII. 

ACTINOMYCOSIS   AND   ALLIED   DISEASES. 

Characters  of  the  actinomyces  —  Tissue  lesions —Distribution  of 
lesions — Cultivation  of  actinomyces — Varieties  of  actinomyces 
and  allied  forms  —  Experimental  inoculation  —  Methods  of 
examination  and  diagnosis — Madura  disease  .  .  .317 


CHAPTER   XIV. 
ANTHRAX. 

Historical  summary — Bacillus  anthracis — Appearances  of  cultures 
— Biology  —  Sporulation — Natural  anthrax  in  animals — Ex- 
perimental anthrax — Anthrax  in  man— Pathology — Toxins  of 
the  bacillus  anthracis — Mode  of  spread  in  nature — Immunisa- 
tion of  animals  against  anthrax — Methods  of  examination  .  331 


CHAPTER  XV. 

TYPHOID  FEVER — BACILLI  ALLIED  TO  THE  TYPHOID  BACILLUS. 

Introductory  —  Bacillus  coli  communis  —  Culture  reactions  — 
Isolation  and  recognition  of  B.  coli — Pathogenic  properties — 
Bacillus  typhosus — Isolation  uud  appearances  of  cultures  — 


CONTENTS  xiii 


reactions  —  Pathological  changes  in  typhoid  fever  — 
Immunisation  of  animals  —  Etiological  relationships  of  bacillus 
typhosus  —  Epidemiology  of  typhoid  fever  —  Typhoid  carriers 
—  Serum  diagnosis  of  typhoid  fever  —  Vaccination  against 
typhoid  —  Methods  of  examination  —  Paratyphoid  and  food- 
poisoning  bacilli  —  The  paratyphoid  bacillus  —  Bacillus  enteri- 
tidis  (Gaertner)  —  The  psittacosis  bacillus  —  Danysz's  bacillus 
and  rat  viruses—  Bacillus  dysenterise  —  Bacillus  enteritidis 
sporogenes  —  Summer  diarrhoea  —  General  review  of  coli-typhoid 
group  ........  350 


CHAPTER  XVI. 
DIPHTHERIA. 

Historical  —  General  facts  —  Bacillus  diphtherire  —  Microscopical 
characters — Distribution — Association  with  other  organisms 
— Cultivation — Powers  of  resistance — Inoculation  experiments 
— The  toxins  of  diphtheria — Variations  in  virulence  of  bacilli — 
Bacilli  allied  to  the  diphtheria  bacillus — Summary  of  patho- 
genic action— Methods  of  diagnosis  ....  396 

CHAPTER  XVII. 

TETANUS. 

Introductory — Historical  —  Bacillus  tetani  —  Isolation  of  bacillus 
tetani — Characters  of  cultures — Conditions  of  growth — Patho- 
genic effects — Experimental  inoculation — Tetanus  toxins  — 
Antitetanic  serum  —  Methods  of  examination  —  Malignant 
( edema — Characters  of  bacillus — Experimental  inoculation — 
Methods  of  diagnosis  —  Bacillus  botulinus  —  Quarter-evil  — 
Bacillus  aerogenes  capsulatus — Fusiform  anaerobic  bacilli  .  415 


CHAPTER  XVIII. 
CHOLERA. 

Introductory — The  cholera  spirillum — Distribution  of  the  spirilla — 
Cultivation — Powers  of  resistance — Experimental  inoculation 
— Toxins  of  cholera  spirillum — Inoculation  of  human  subject 
—  Immunity  —  Methods  of  diagnosis  —  General  summary  — 
Other  spirilla  resembling  the  cholera  organism — Metchnikoflf's 
spirillum — Finkler  and  Prior's  spirillum — Deneke's  spirillum  446 


xiv  CONTENTS 

CHAPTER  XIX. 

INFLUENZA,  WHOOPING-COUGH,  PLAGUE,  MALTA  FEVER. 

PAGE 

Influenza  bacillus — Microscopical  characters — Cultivation  —  Dis- 
tribution—  Experimental  inoculation — Methods  .of  examina- 
tion—  Whooping-cough  bacillus — Microscopical  characters  — 
Pathogenic  effects  —  Methods  of  examination  —  Bacillus  of 
plague  —  Microscopical  characters  —  Cultivation  —  Anatomical 
changes  produced  and  distribution  of  bacilli — Experimental 
inoculation — Paths  and  mode  of  infection — Toxins,  immunity, 
etc. — Preventive  inoculation — Anti-plague  sera — Methods  of 
diagnosis — Malta  fever — Micrococcus  melitensis — Relations  to 
the  disease  —  Mode  of  spread  of  the  disease  —  Methods  of 
diagnosis  .  .  .  .  .  .  .  467 

CHAPTER  XX. 

DISEASES  DUE  TO  SPIROCH^TES — THE  RELAPSING  FEVERS, 
SYPHILIS,  AND  FRAMBCESIA. 

Relapsing  fever  and  African  tick  fever — Characters  of  the  spirochsete 
— Relations  to  the  disease — Immunity — African  tick  fever — 
Transmission  of  the  disease— Syphilis — Microscopic  characters 
of  spirochsete  pallida  —  Distribution  —  Cultivation  —  Trans- 
mission of  the  disease --Serum  Diagnosis  —  Wassermann 
reaction — Frambcesia  or  Yaws  .  494 


CHAPTER  XXI. 
IMMUNITY. 

Introductory  —  Acquired  immunity  —  Artificial  immunity  — 
Varieties  —  Active  immunity  —  Methods  of  production  —  At- 
tenuation and  exaltation  of  virulence — Passive  immunity — 
Action  of  the  serum  —  Antitoxic  serum  —  Standardising  of 
toxins  and  of  anti-sera — Nature  of  antitoxic  action — Ehrlich's 
theory  of  the  constitution  of  toxins — Antibacterial  serum — 
Bactericidal  and  lysogenic  action  —  Hsemolytic  and  other 
sera — Methods  of  the  hsemoly tic  tests — Opsonic  action — Ag- 
glutination—  Precipitins  —  Therapeutic  effects  of  anti-sera  — 
Theories  as  to  acquired  immunity — Ehrlich's  side-chain  theory 
—  Theory  of  phagocytosis  - —  Natural  immunity  —  Natural 
bactericidal  powers  —  Natural  susceptibility  to  toxins  — 
Supersensitiveness  or  anaphylaxis — The  serum  disease  in  man  512 


CONTENTS  xv 

APPENDIX  A. 

SMALLPOX  AND  VACCINATION. 

PAGE 

.Irnnerian  vaccination  —  Relationship  of  smallpox  to  cowpox  — 
Micro-organisms  associated  with  smallpox — The  nature  of 
vaccination  .  .  .  .  .  .  .  565 


APPENDIX  B. 
HYDROPHOBIA. 

Introductory— Pathology — The  virus  of  hydrophobia— Prophylaxis 

— Antirabic  serum — Methods    .  .       573 


APPENDIX   C. 
MALARIAL  FEVER. 

The  malarial  parasite — The  cycle  of  the  malarial  parasite  in  man 
— The  cycle  in  the  mosquito — Varieties  of  the  malarial  para- 
site—  General  considerations  —  The  pathology  of  malaria  — 
Methods  of  examination  .  .  585 


APPENDIX  D. 

AMOSBIC  DYSENTERY. 

AniM-bic  dysentery — Characters  of  the  amoeba — Cultivation  of  the 
aiiM'bji'-  Distribution  of  the  amoebse — Experimental  inocula- 
tion— Methods  of  examination  ...  .  G02 


APPENDIX   E. 

TRYPANOSOMIASIS — LEISHMANIOSLS — PIROPLASMOSIS. 

The  pathogenic  trypanosomes  —  Morphology  and  biology  of  the 
trypanosomata  — Trypanosoma  Lewisi— Nagana  or  tse-tse  fly 
disease  —  Trypanosome  of  sleeping  sickness  —  Trypanosoma 
Cruzi  —  Leishmaniosis  —  Leishmania  Donovani  —  Leishmania 
infantum  — Leishmania  tropica  —  Histoplasma  capsulatum  — 

.  .  .  .  .  .  .610 


xvi  CONTENTS 

APPENDIX   F. 

T7.  PAGE 

YELLOW  FEVER     ....  g39 

APPENDIX  G. 

ACUTE  POLIOMYELITIS      .  .  544 

APPENDIX   H. 
PHLEBOTOMUS  FEVER  •     .  .  g46 

APPENDIX  J. 

TYPHUS  FEVER      .  .  .  g48 

BIBLIOGRAPHY        .  .  649 

INDEX  .  673 


LIST  OF  COLOURED   PLATES. 


PLATE  I. 
fa. 

1.  Film  of  pus,  containing  staphylococci  and  streptococci. 

2.  Fraenkel's  pneumococcus  in  sputum. 

.'>.   Meningococcus  in  epidemic  cerebro-spinal  fever. 

4.  Film  from  a  scraping  of  throat  in  Vincent's  angina,  showing  fusiform 

bacilli  and  spirocluetes. 

5.  Gouorrhoeal  pus,  showing  gonococci  and  staphylococci. 

PLATE  II. 

6.  Spirochaete  pallida,  case  of  congenital  syphilis. 

7.  Tubercle  bacillus  and  other  bacteria  in  sputum. 

8.  Leprous  skin,  showing  clumps  of  bacilli  in  the  cutis. 

9.  Leprous  granulation  tissue,  showing  bacilli. 

PLATE  III. 

10.  Stivptothrix  actinomyces. 

11.  Anthrax  bacilli. 

12.  Bacillus  diphtheria;. 

13.  Bacillus  diphtheria  (involution  forms). 
1  l.  Ilofmann's  pseudo-diphtheria  bacillus. 

15.  Typhoid  bacillus,  showing  flagella. 

xvii 


xviii  LIST  OF  COLOURED  PLATES 

PLATE  IV. 

FIG. 

16.  Negri  bodies  in  nerve  cells  in  rabies. 

17.  Bacillus  pestis  (involution  forms). 

18.  Spirochaete  of  relapsing  fever. 

19.  The  cholera  spirillum,  showing  Hagella. 

20.  Bacillus  tetani,  showing  spores. 

PLATE  V. 

21.  The  Parasite  of  Mild  Tertian  Malaria. 

Cycle    I.  (Schizogony).     Asexual  cycle  in  the  human  blood. 
Cycle  II.  (Sporogony).     Sexual  cycle  in  the  mosquito. 

22.  The  Parasite  of  Malignant  Malaria. 

PLATE  VI. 

23.  Entamoeba  histolytica  in  pus,  from  tropical  abscess  of  liver. 

24.  Leishman-Donovau  bodies,  from  a  case  of  Kala-azar. 

25.  Trypanosoma  Gambiense. 


LIST  OF  ILLUSTRATIONS  IN  TEXT. 


FIO.  PAGE 

1.  Forms  of  bacteria            .             .             .                          .             .13 

•_'.  I  lot-air  steriliser                          ...                         .28 

:}.  Koch's  steam  steriliser  .             .             .             .                                   28 

4.  Autoclave  .             .             .             .             .             .             .30 

5.  Steriliser  for  blood  serum  .             .            .             .             .31 

6.  Meat  press  .......         32 

7.  Hot-water  funnel  ......         36 

8.  Blood  serum  inspissator  .             .             .  •           .             .41 

9.  Potato  jar  .             .                                        .             .         45 

10.  Cylinder  of  potato  cut  obliquely  .  .  .  .45 

11.  Ehrlich's  tube  containing  piece  of  potato  .         •    .  .46 

12.  Apparatus  for  filling  tubes        '\  .  .  .  .53 

13.  Tubes  of  media  .  .  .  .  .  .53 

14.  Platinum  wires  in  glass  handles  .  .  .  .54 
I."-.  Method  of  inoculating  solid  tubes          ....         55 

16.  Rack  for  platinum  needles          .....         56 

17.  Petri's  capsule     .  .  .  .  .  .  .         57 

1  x.    Koch's  levelling  apparatus  for  use  in  preparing  plates  .  .         58 

19.  Koch's  levelling  apparatus         .             .             .             .  .58 

20.  Esmarch's  tube  for  roll  culture  .             ...            .  .60 

21.  Apparatus  for  supplying  hydrogen  for  anaerobic  cultures  .         63 

22.  Esmarch's  roll-tube  adapted  for  culture  containing  anaerobes  .         64 

23.  Bulloch's  apparatus  for  anaerobic  plate  cultures            .  .64 

24.  Flask  for  anaerobes  in  liquid  media       .             .             .  .67 

25.  Flask  arranged  for  culture  of  anaerobes  which  develop  gas  .         68 

26.  Tubes  for  anaerobic  cultures  on  the  surface  of  solid  media  .         68 

27.  Slides  for  hanging-drop  cultures             .            .            .  .69 

28.  Apparatus  for  counting  colonies             .             .             .  .70 

29.  Wright's  250  c.mm.  pipette  fitted  with  nipple              .  .         71 

30.  Geissler's  vacuum  pump  for  filtering  cultures    .  75 

31.  Chamberland's  candle  and  flask  arranged  for  filtration  .         75 
:;±  < ,'h  ^label-land's  bougie  with  lamp  funnel            .            ...         76 


xx  LIST  OF  ILLUSTRATIONS  IN  TEXT 

FIG.  PAGE 

33.  Bougie  inserted  through  rubber  stopper  .  .  .76 

34.  Muencke's  modification  of  Chamberland's  filter             .             .  77 

35.  Flask  for  filtering  small  quantities  of  fluid        .  78 

36.  Tubes  for  demonstrating  gas-formation  by  bacteria       .             .  81 

37.  Geryk  air-pump  for  drying  in  vacua      .  .85 

38.  Reichert's  gas  regulator .  .  .  .  .86 

39.  Hearson's  incubator  for  use  at  37°  C.      .             .             .             .  87 

40.  Cornet's  forceps  for  holding  cover-glasses  .  .  .94 

41.  Needle  with  square  of  paper  on  end  for  manipulating  paraffin 

sections           .             .             .             .            .  99 

42.  Syphon  wash-bottle  for  distilled  water  .  102 

43.  Wright's  5  c. mm.  pipette         ".             .             .             .          .  .  118 

44.  Tubes  used  in  testing  agglutinating  and  sedimenting  properties 

of  serum         .            .            .            .            .                        ;  119 

45.  Wright's  blood-capsule  .             .             .             .  124 

46.  Test-tube  and  pipette  arranged  for  obtaining  fluids  .containing 

bacteria  .  "..  .  .  ,:  -          .  .136 

47.  Hollow  needle  for  intraperitoneal  inoculations  .           ..   ':'         .  143 

48.  Hesse's  tube        .             .             .             .    •     .    .                     j - .  1 48 

49.  Petal's  sand  filter            .                          .                                       .  149 

50.  Staphylococcus    pyogenes    aureus,    young    culture    on    agar. 

xlOOO  ....  .  ,203 

51.  Two  stab  cultures  of  Staphylococcus  pyogenes  aureus  in  gelatin  203 

52.  Streptococcus  pyogenes,  young  culture  on  agar.      x  1000        ;»  204 

53.  Culture  of  the  streptococcus  pyogenes  on  an  agar  plate          ji^-  205 

54.  Bacillus  pyocyaneus  ;  young  culture  on  agar.      x  1000              I'  208 

55.  Micrococcus  tetragenus.      x  1000           .             .             .         -."...  209 

56.  Streptococci  in  acute  suppuration,      x  1000       .             .             .  212 

57.  Minute  focus  of  commencing  suppuration  in  brain,      x  50         .  214 

58.  Secondary  infection  of  a  glomerulus  of  kidney  by  the  Staphylo- 

coccus aureus.      x 300            ...             .             .             .  215 

59.  Section  of  a  vegetation  in  ulcerative  endocarditis,      x  600        .  217 

60.  Film  preparation  from  a  case  of  acute  conjunctivitis,  showing 

the  Koch-Weeks  bacilli,      x  1000     .....  219 

61.  Film  preparation  of  conjunctival  secretion  showing  the  diplo- 

bacillus  of  conjunctivitis,      x  1000    .             .             .          -.  220 

62.  Film  preparation  of  pneumonic  sputum,   showing   numerous 

pneumococci  (Fraenkel's).      x  1000   .             .             .           '  .  227 

63.  Friedlander's    pneumobacillus,    from    exudate    in    a    case    of 

pneumonia,      x  1000 .             „  •                       .             .             .  228 

64.  Fraenkel's  pneumococcus  in  serous  exudation,      x  1000             .  228 

65.  Stroke  culture  of  Fraenkel's  pneumococcus  on  blood  agar          .  229 

66.  Fraenkel's  pneumococcus  from  a  pure  culture  on  blood  agar. 

xlOOO  .  .  .  .  .  .  .230 

67.  Stab  culture  of  Friedlander's  pneumobacillus    .,-.-,             .  232 


LIST  OF  ILLUSTRATIONS  IN  TEXT  xxi 

FIG.  PAGE 

68.  Friedliinder's  pneumobacillus,  from  a  young  culture  on  agar. 

xlOOO            .                                                                             .  233 

69.  Capsulated  pneumococci  in  blood  taken  from  the  heart  of  a 

rabbit.      x  1000        ...                                        .  236 

70.  Film  preparation  of  exudation   from   a   case  of  meningitis. 

xlOOO           .                                                                                .  242 

71.  Pure  culture  of  diplococcus  intracellulai  is        .             .             .  243 

72.  Portion  of  film  of  gonorrhceal  pus.      x  1000     .  250 

73.  Colonies  of  gouococcus  on  serum  agar  .             .                          .  251 

74.  Gonococci,  from  a  pure  culture  on  blood  agar.      x  1000           .  251 

75.  Film  preparations  of  pus  from  soft  chancre,  showing  Ducrey'.s 

bacillus.      x!500     ....  .258 

76.  Ducrey'.s  bacillus     x  1500         .                                                    .  259 

77.  Tubercle  bacilli,  from  a  pure  culture  on  glycerin  agar.      x  1000  262 

78.  Tubercle  bacilli  in  phthisical  sputum,      x  1000  263 

79.  Cultures  of  tubercle  bacilli  on  glycerin  agar     .             .             .  266 
"80.  Tubercle  bacilli  in  section  of  human  lung  in  acute  phthisis. 

xlOOO  .....  .270 

81.  Tubercle  bacilli  in  giant-cells,      x  1000           .             .             .  271 

82.  Tubercle  bacilli  in  urine,      x  1000       .            .                         .  272 

83.  Hoeller's  Timothy-grass  bacillus,      x  1000       .  .279 

84.  Cultures  of  acid-fast  bacilli  grown  at  room  temperature           .  279 

85.  Smegma  bacilli,      x  1000          .  .  .  .280 

86.  Section  through  leprous  skin,  showing  the  masses  of  cellular 

granulation  tissue  in  the  cutis.      x  80            .             .             .  298 

S7.  Superficial  part  of  leprous  skin,      x  500           .                          .  300 

88.  High-power  view  of  portion  of  leprous  nodule  showing  the 

arrangement  of  the  bacilli  within  the  cells  of  the  granula- 
tion tissue,      x  1100              .....  301 

89.  Glanders  bacilli  from  peritoneal  exudate  of  guinea-pig,     x  1000  308 

90.  Glanders  bacilli,      x  1000         .                                                    .  309 

91.  Actinomycosis  of  human  liver,      x  500            .                          .  319 

92.  Actinomyces  in  human  kidney,      x  500           .             .             .  320 

93.  Colonies  of  actinomyces.      x  60             .             .             .        ->•-<.  321 

94.  Cultures  of  the  actinomyces  on  glycerin  agar.      x  60  .             .  324 

95.  Actinomyces,  from  a  culture  on  glycerin  agar.      x  1000          .  325 

96.  Shake  cultures  of  actinomyces  in  glucose  agar.                          .  •  326 

97.  Section  of  a  colony  of  actinomyces  from  a  culture  in  blood 

serum,      x  1500          .            .            .            .            .            .  326 

98.  Streptothrix  Madura,      x  1000.  .  .  .  .329 

99.  Surface  colony  of  the   anthrax  bacillus   on  an  agar  plate. 

x30.  .  .  .  .  .  .333 

100.  Anthrax   bacilli,    arranged    in   chains,    from    a   twenty-four 

hours'  culture  on  agar  at  37°  C.      x  1000     .             .             .  334 

101.  Stab  culture  of  the  anthrax  bacillus  in  peptone-gelatin            .  334 


xxil  LIST  OF  ILLUSTRATIONS  IN  TEXT 

FIG.  PAGE 

102.  Anthrax  bacilli  containing  spores,      x  1000     .                          .  336 

103.  Scraping  from  spleen  of  guinea-pig  dead  of  anthrax.       x  1000  339 

104.  Portion  of  kidney  of  a  guinea-pig  dead  of  anthrax,      x  300    .'  340 

105.  Bacillus  coli  communis.      xlOOO         .           •  '.-.           .            -...  351 

106.  A  large  clump  of  typhoid  bacilli  in  a  spleen,      x  500  .             .  357 

107.  Typhoid  bacilli,  from  a  young  culture  on  agar,  showing  some 

filamentous  forms.      xlOOO.             ....  358 

108.  Typhoid  bacilli,    from   a   young  culture   on  agar,   showing 

flagella.  .  x  1000      ....  .359 

109.  Culture  of  the  typhoid  bacillus  and  of  the  bacillus  coli            .  360 

110.  Colonies  of  the  typhoid  bacillus  in  a  gelatin  plate,      x  15       .  361 

111.  Film    preparation    from    diphtheria    membrane  ;     showing 

numerous  diphtheria  bacilli,      x  1000                       .             .  398 

112.  Section  through  a  diphtheritic  membrane  in  trachea,  show- 

ing diphtheria  bacilli.      xlOOO        .             .             .  399 

113.  Cultures  of  the  diphtheria  bacillus  on  an  agar  plate    .         .    .  401 

114.  Diphtheria  colonies,  two  days  old,  on  agar.      x  8        .         .    .  401 

115.  Diphtheria    bacilli    from    a    twenty-four    hours'   culture   ou 

agar.      x  1000           .....             .  402 

116.  Diphtheria  bacilli,  from  a  three  days'  agar  culture,      x  1000  .  402 

117.  Involution  forms  of  the  diphtheria  bacillus.      xlOOO.          •••..'•-  403 

118.  Pseudo-diphtheria  bacillus  (Hofmann's).      x  1000       .         ,    ,,  411 

119.  Xerosis  bacillus  from  a  young  agar  culture,      x  1000  .             .  412 

120.  Film    preparation   of  discharge    from   wound   in    a   case    of 

tetanus,  showing  several  tetanus  bacilli  of  "drumstick" 

form.      xlOOO          ......  417 

121.  Tetanus  bacilli,  showing  flagella.      x  1000      .             .             .  418 

122.  Spiral  composed  of  numerous  twisted  flagella  of  the  tetanus 

bacillus.      xlOOO     ...  .419 

123.  Tetanus  bacilli,  some  of  which  possess  spores,      x  1000           .  419 

124.  Stab  culture  of  the  tetanus  bacillus  in  glucose  gelatin             .  420 

125.  Colonies  of  the  tetanus  bacillus  on  agar  seven  days  old.      x  50  421 

126.  Film    preparation    from    the    affected    tissues    in    a    case    of 

malignant  oedema.      x  1000               .             .                           •  434 

127.  Bacillus  of  malignant  cedcma,  showing  spores,      x  1000          .  435 

128.  Stab  cultures  in  agar — tetanus  bacillus,  bacillus  of  malignant 

oedema,  and  bacillus  of  quarter-evil             .             .             .  436 

129.  Bacillus  of  quarter-evil,  showing  spores.      x  1000        .             .  442 

130.  Bacillus  aerogenes  capsulatus    .....  443 

131.  Cholera  spirilla,  from  a  culture  on  agar  of  twenty-four  hours' 

growth.      xlOOO      ....  .447 

132.  Cholera    spirilla    stained     to    show    the    terminal    flagella. 

xlOOO  ....  .448 

133.  Cholera  spirilla  from  an  old  agar  culture.      x  1000      .              .  448 

134.  Puncture  culture  of  the  cholera  spirillum         .             .             .  450 


LIST  OF   ILLUSTRATIONS  IN  TKXT  xxiii 

Hi;.  PACK 

135.  Colonies  of  the  cholera  spirillum  on  a  gelatin  plate     .             .  451 

136.  MetchnikoflTs  spirillum,      x  1000  .  .  .464 

137.  Puncture  cultures  in  peptone-gelatin  ....  465 

138.  Finkler  and  Prior's  spirillum,      x  1000            .             .             .  466 

139.  Influenza  bacilli  from  a  culture  on  blood  agar.      x  1000          .  467 

140.  Film  preparation  from  a  twenty-four  hours'  culture  of  the 

whooping-cough  bacillus.      x  1000  ....  473 

141.  Film  preparation  from  a  plague  bubo.      x  1000            .             .  476 

142.  Bacillus  of  plague  from  a  young  culture  on  agar.      x  1000      .  477 

143.  Bacillus  of  plague  in  chains,      x  1000  .             .             .             .  477 

144.  Culture  of  the  bacillus  of  plague  on  4  per  cent,  salt  agar. 

xlOOO          .......  478 

145.  Section  of  a  human  lymphatic  gland  in  plague,      x  50            .  480 

146.  Film  preparation  of  spleen  of  rat  after  inoculation  with  the 

bacillus  of  plague,      x  1000 .             ....  482 

!  17.   Mirrococrus  melitensis.      x  1000          .             .             .             .  490 

148.  Spirilla  of  relapsing  fever  in  human  blood,      x  about  1000     .  496 

149.  Spirillum  Obermeieri  in  blood  of  infected  mouse,      x  1000      .  197 

150.  Film   of  human   blood   containing  spirillum   of  tick  fever. 

x 1000          .......  500 

151.  Spirillum  of  human  tick  fever  (Spirillum  Duttoni)  in  blood 

of  infected  mouse,  x  1000  .  .  .  .  .501 

152  and  153.  Film  preparation  from  juice  of  hard  chancre  showing 

spirochaete  pallida.  x  1000  ....  504 
151.  Film  preparation  from  juice  of  hard  chancre  showing 

spirochaete  pallida.  x  2000  .  .  .  .505 

s.'Ctii.n  of  spleen  from  a  case  of  congenital  syphilis,  showing 

^pirochaete  pallida.  x  1000  ....  506 

15*1.  Spiro"h;i-r«<  refringens.  x  1000  ....  507 

157-162.  Various  phases  of  the  benign  tertian  parasite  .  .  589 

163-168.  Exemplifying  phases  of  the  malignant  parasite  .  .  590 

169.  Anueba? .of dysentery    ......  603 

170.  Section  of  wall  of  liver  abscess,  showing  an  amoeba  of  spherical 

form  with  vacuolated  protoplasm,      x  1000             .            .  607 

171.  Trypanosoma  Brucei  from  blood  of  infected  rat.     Note  in  two 

of  the  organisms  commencing  division  of  micronucleus  and 

undulating  membrane,      x  1000       ....  620 

1  72.  Trypanosoma  gambiense  from  blood  of  guinea-pig,      x  1000  .  623 

173.  Leishmau-Donovan  bodies  from  spleen  smear,      x  1000           .  632 
174    Leishman- Donovan  bodies  within  endothelial  cell  in  spleen. 

xlOOO          ...  633 


•     0  *     "'     *J 

PLATE  I. 


FIG.  1.  Film  of  pus,  containing  staphylococci  and  streptococci.      Stained 
^  .by  Gram's  method.  x  1000  diameters. 

FIG.  2.  Fraenkel's  pneumococcus  in  sputum,  from  a  case  of  acute 
pneumonia.  Rd.  Muir's  method  of  capsule  staining. 

x  1000  diameters. 

Fid.  3.  Meningococcus  in  epidemic  cerebro- spinal  fever,  from  lumbar 
puncture  fluid,  showing  some  involution  forms.  Leishman's 
stain.  x  1000  diameters. 

FIG.  4.  Film  from  a  scraping  of  throat  in  Vincent's  angina,  showing 
fusiform  bacilli  and  spirochaetes.  x  1000  diameters. 

FIG.  5.  Gonorrhceal  pus,  showing  gonococci  (stained  red)  and  staphylo- 
cocci. Gram's  method.  x  1000  diameters. 


.}    tfT/.J'I 

. 


r,    1o    98JSO    &    rnoi 

oiuaqflo  I 

• 
-uf^ilq.f 


PLATE    I. 


r., 

P      ,< 

x 


t 


rtsr  .£ 

••    k 


A 
\ 


FIG.  1. 


FIG.  2. 


FIG.  4. 


FIG.  5. 


.' 


t 


PLATE   II. 


FIG.  6.  Spirochaete  pallida  in  section  of  spleen  of   child;   case   of   con- 
genital syphilis.     Levaditi's  stain.  x  1000  diameters. 


FIG.  7.  Tubercle  bacillus  and  other  bacteria  in  sputum  ;  case  of  chronic 
phthisis.     Ziehl-Neelsen  stain.  x  1000  diameters. 


FIG.  8.  Section  of  leprous  skin,  showing  numerous  clumps  of  bacilli 
(stained  red)  in  the  cutis.  Carbol-fuchsin  and  methylene- 
blue.  x  80  diameters. 

FIG.  9.  Section  of  leprous  granulation  tissue,  showing  large  numbers  of 
bacilli,  chiefly  contained  within  cells.  Carbol-fuchsin  and 
methylene-blue.  x  1000  diameters. 


FIG.  8.  Fio.  9. 


.II   HTAJ'I 

-noo   lo   saeo   ;  blrrlo   lo  aaalfp.   ^<>  n«\:  ''ii 

0001  x  .m-fite  a'idib^vaJ     .  'i  ^nag 


oinoirfo  lo  ea^o  ;  mw^uqa  nr  fiheto&if  isriJo  bnc  «rj[fioed  oIoioduT  .7  . 
,r;ib  0001  x  .nw:' 


lo   «qnrufo   guoiomnn    gniv  .8  .oil 

ij^-lodieO      .aijuu   edi   at 

('i3  x 

.^j  srroiqal  .8  .aiU 

;ri-l<jdijs0      .p.IIao    nirii///    b 

.ankf 


PLATE    II. 


FIG.  6 


FIG.  7. 


FIG.  8. 


FIG.  9. 


•• 

•  1 


PLATE  III. 


FIG.  10.  Streptothrix  actinomyces,  from  agar  culture.     Gram's  method. 

x  1000  diameters. 

FIG.  11.  Anthrax  bacilli,    from    4-days'    agar  culture,    showing  spores. 
Carbol-fuchsin  and  methylene-blue.  x  1000  diameters. 

FIG.  12.  Bacillus   diphtherias,    from    a    12-houre'    blood    serum   culture. 
Neisser's  stain  modified.  x  1000  diameters. 

N\V" 

FIG.  13.   Bacillus  diphtheria;,  from  a  5-days'  blood  serum  culture,  show- 
ing involution  forms.     Neisser's  stain  modified. 

x  1000  diameters. 

FIG.  14.  Pseudo-diphtheria    bacillus     (Hofmann's),    from    young    agar 
culture.      Neisser's  stain  modified.  x  1000  diameters. 


FIG.  15.  Typhoid    bacillus,    from    a    24-hours'    agar    culture,    showing 
flagella.     Rd.  Muir's  method.  x  1000  diameters. 


* 


!» 


+'    \ 


u 


V 


Fir,.  15. 


.Ill  3TAJ1 

.bodtem  g'mjsir)     .aiuiltio  ifigB  mail  ,890^moniio«  zhtfoodqeiitB  .01  .oil 


.e.8-£oqa  §niwods    piuiiuo  ia%s   (ey&b-l    moil    ,'rflioad  xeidinA  .ft  .oil 
fb  0001  x  .auld-anafyfoem  baa  fli 


.9«rtluo   inow?.    boold    'eiwod-SI    *    moil    .aBnadidqil)  80IUo«S  .SI  . 
sib  0001  x  .bsftibooi  areda 


-woria  ,9iuiluo  minsa  faoofcf  'e^fi{)-s 

.baftibom  nr«i«  s'ldaaisll     .amiol  noidulovni  gni 
ooiBib  0001  x 


moil    ,(8'nn«raloH)     aaHioad     B 
x  .baftibora  nifiie  •  a^aaeisVI      , 


jiniwoda    piuiluo    IB§*    ^uod-^S     fi     raoil    ,ei/IIioud    biodcftT  .d[  . 
tjiib  0001  x  .boriiara  a'liwM  .MI 


PLATP]    III. 


-     • 


"• 


•    •  %  • f  v  : 

•    » •        •  •  9 1 


FIG.  10. 


FIG.  11. 


.  /. 


•  .  >•' 


»..*.»     * 
•  *»  * 

*    r     •  * 

!•    *     • 


FIG.   12. 


Fir;.   13. 


:-4);^ 

'  V '•'-•%?>-' 
',;  N-.  - 1  '(31/ , 

'  '..'v  .0— T 


\ 


FIG.  14. 


FIG.   15. 


PLATE  IV. 


FIG.  16.  Negri   bodies  in  nerve   cells  in   rabies   (hippocampus  of  dog). 
Alcoholic  eoein  and  methylene-blue.  x  1000  diameters. 

FIG.  17.  Bacillus  pestis,    showing    involution    forms,    from    a    salt-agar 
culture.  x  1000  diameters. 

FIG.  18.  Blood     film,     showing    the    spirochaete    of     relapsing    fever. 
Irishman's  stain.  x  1000  diameters. 

FIG.  19.  The  cholera  spirillum,  from  a  12-hours'  agar  culture,  showing 
flagella.  x  1000  diameters. 

FIG.  20.  Bacillus  tetani,  showing  spores.  x  1000  diameters. 


Km.   19. 


20 


.VI   ttT/.I'i 


.(gob   to   auqraaooqqrd)   aaidfii   ni   allao   svtsa  ni  aeibod   iiga^l   .81  .oil 
ib  0001  x  .sufd-anatyrftem  bna  niaoa  oiforiooIA 

moil    ,8miol    noidulovai    §aiworfp.    tai 


x 


.19Y91    ^nieqfilai     to     sdasffooiiqa     9fld     gniworfa     (inlft     b«x)fS[   .81  .oi' 
.ai*tem«ib  0001  x  .nicia  8'nBcari«i9J 


,9iuiloo  i*^«  '«iuorf-^I   «  moil  ,ranllhiq8  fiialorfo  odT  ,61  .01*? 
ib  0001  x 


.aieianwib  0001  x  .gb'ioqa  ^niworie  ,in«l9i  aullio«a  .OS  .01 


PLATE    IV. 


Fio.  16. 


s 


c^ 

<J^ 


FIG. -18. 


<       ' 
f 


J_ 
FIG.  19. 


FIG.  17. 


_--y 

\    OX    \ 


JO  1 


-  ^     S 


N  / 


i. 


FIG.  20. 


ATE    V. 

PLATE  V. 

FIG.  21.  THE  PARASITE  OP  MILD  TERTIAN  MALARIA. 
Cycle  I.  (Schizogony).     Asexual  cycle  in  the  human  blood. 

a.    Sporozoite    entering    red     blood     corpuscle    and     forming 

young  trophozoite. 
6.    Young  trophozoite  in  red  blood  corpuscle. 

c.  Young  trophozoite  in  red  blood  corpuscle,  with  accumulation 

of  pigment. 

d.  Large  pigmented  trophozoite. 
' .    Mature  schizont. 

/.  Commencing  segmentation  of  schizont. 

g.  Further  stage  of  segmentation. 

h.  Segmented  schizont ;  formation  of  merozoites. 

i.  Disintegration   of    red    blood    corpuscle,    setting    free    the 

merozoites. 

j.  Young  merozoite  entering  red  blood  corpuscle. 

k.  Macrogametocyte,  or  female  sporont. 

I.  Microgametocyte,  or  male  sporont. 

Cycle  II.  (Sporogony).     Sexual  cycle  in  the  mosquito, 

'ra.  Microgametocyte. 

n.  Macrogametocyte. 

Formation  of  microgametes  from  the  microgametocyte. 

p.  Free  microgamete. 

q.  Microgamete  entering  the  macrogametocyte. 

jr.  Zygote  or  ookinete. 

s.  Sporocyst. 

t.  Formation  of  sporoblasts  in  the  sporocyst. 

u.  Formation  of  sporozoites  from  sporoblasts. 

v.  Rupture  of  sporocyst,  setting  free  the  sporozoites. 

to.  Free  sporozoites  in  the  body  fluid. 

i.  Accumulation  of  sporozoites  in  the  salivary  gland. 

y.  Sporozoites  passing  from  gland  duct  into  the  blood  of  man. 

FIG.  22.  THE  PARASITE  OF  MALIGNANT  MALARIA. 

a.    Young  trophozoite  entering  red  blood  corpuscle. 
6.  Do.  in  red  corpuscle. 

c.  Multiple  infection  of  red  corpuscle. 

f/.  Multiple  infection  with  chromatic  stippling  in  cellular  proto- 
plasm ;  a  similar  cell  is  seen  lying  beneath  a, — it  contains 
a  pigmented  trophozoite. 

d.  Pigmented  trophozoite. 

e.  Segmented  schizont,  cluster  of  merozoites. 
/.    Macrogametocyte,  "  female  crescent." 

g.    Microgametocyte,  "  male  crescent." 

A.    Red  blood  corpuscle  with  chromatic  stippling. 

i.    Large  mono-nucleated  phagocyte  containing  malarial  pigment 


.V 

.AI&AJ&M  viAixaaT  aaiM  *o  HTIHAHAI  auT  .IS  . 
.boold  a&mud  ad*  m  alo^o  Jtor/xseA 
ytinnol     bn*    alosi/q-ioo     boold     bai    gnhsJna 

.eiioxodqoiit 

.sloeuqioo  boold  bsi  ni  aJioxoriqoi;* 
,9loeuqTOo  boold  bei  ni  atiosodqoTi 


.inosidoa  to  noritfiJnam^aB  gnionaraaioO    .\ 


to  noiteanoi  ;  inoxidoe  boin^jiryy^    .A 
91    io    n(  'L    '* 


sdi  gahsiaa  9^ara«yrwiM    .? 
10  aio^iS    .*» 


ni  o»a«ldoioqa  to  noiteonol     .S 
moil  gsiiosoioqa  to  noii«flno^    .u 
edi  aai^  ^niiiaa  ^a^ooioqe  to  artrtqw.fi    .« 


bai  §flho*£ia  aiiosoiaoi 

10  , 

.*flOioqa  alam  10 
.oJiupeoia  ad*  ni  alo^0  I*ff»8     .(^nogoioqfc: 

"  '£ 
a*'£>o*i 
moil  •ttMMBMobi  to  noiiaanol    .o 


£•  !•  ^ 

s*6"  ^ 


.bflfils  ^wvilea  ad*  ni  aa*ioso'ioqa  to  nohtalimiuosA  .x 

.item  to  boold  ad*  o*ar  *oub  bnalg  moi!  ^aiaaaq  aa*iosoioq8  .yj 

.AIHAJAM  TWAMOIJAM  TO  STiaAflAl   SHT    .SS  .Ol' 

.afoaifqioo  boold  bai  gniiaixia  e^iosodqoi*  gnnox  .» 

.aloai/qioo  bai  ai  -^. 

.aloe/jqioo  bai  to  n<'  -Ii/M  .a 

-oioiq  lalullao  ai  gnilqqiia  oiJamoirio  ri*iw  a-  r~  •  'J 

ii (0  d*aanad  ^ni^I  naaa  ei  flao  lalioiie  a  ;  aiafiiq 

.ajiosodqoi*  ba*aaui^iq  a 

,ajios(;dqoi*  baiaaoi^i*!  .b 
tinosidoa  h9*aama98  .^> 
J'  ,a*^oo*aina^oio«M  .\ 

ta*^U'  «  ^1 

Iqqi*8  ocJamoirio  d*tw  afoeuqioo  «A 
sfli^iq  Iciialam  §nini**noo  a^xoogailq  ba*aaio0fl-onom  agiaJ     .1 


PLATE    V. 
FIG.  21. 


-fc  &  ^ 


FIG.   22. 


PLATE   VI. 


FIG.  23.  Entamceba   histolytica  in  pus,   from   tropical   abscess  of  liver. 

»Wet  fixed  film.      Stained  by  Benda's  method, 
x  1000  diameters. 

Fio.  24.    Leishman-Donovan    bodies,    from    the    spleen    of    a    case    of 
ir.  x  1000  diameters. 


FIG.  25.  Blood  film,  showing  Trypanosoma  Gambiense.    Irishman's  stain. 

x  1000  diameters. 


.IV 


.lav  il   to  839.owte   koiqoiJ   11101}    ,*rrq  ni  aoiityloieuf   «da>raj8^na  .8S  .oil 

.b 
rh  000  1  x 


lo    9fMjo    «    to    n 
iib  0001  x 


.ni*ig  g'luoiffaisJ    .saasidmAf)  AIHOK-  •  woria  .ralS  b<x)ia  .SS  .oil 

OOOf  x 


PLATE  ;yi 


Fia.  23. 


FIG.  24. 


I 


FIG.  25. 


MANUAL  OF  BACTERIOLOGY 

CHAPTER   I. 

GENERAL  MORPHOLOGY  AND  BIOLOGY. 

Introductory. — At  the  bottom  of  the  scale  of  living  things  there 
exists  a  group  of  organisms  to  which  the  name  of  bacteria  is 
usually  applied.  These  are  apparently  of  very  simple  structure, 
and  may  l>e  subdivided  into  two  sub-groups,  a  lower  and'simpler 
and  a  higher  and  better-developed. 

The  lower  forms  are  the  more  numerous,  and  consist  of 
minute  unicellular  masses  of  protoplasm  devoid  of  chlorophyll, 
which  multiply  by  simple  fission.  Some  are  motile,  others  non- 
motile.  Their  minuteness  may  be  judged  of  by  the  fact  that  in 
one  direction  at  least  they  usually  do  not  measure  more  than 
1  n,  (^-Tfjffir  inch).  These  forms  can  be  classified  according  to 
their  shapes  into  three  main  groups — (1)  A  group  in  which  the 
shape  is  globular.  The  members  of  this  are  called  cocci.  (2)  A 
group  in  which  the  shape  is  that  of  a  straight  rod — the  pro- 
portion of  the  length  to  the  breadth  of  the  rod  varying  greatly 
among  the  different  members.  These  are  called  bacilli.  (3)  A 
group  in  which  the  shape  is  that  of  a  curved  or  spiral  rod. 
These  are  called  spirilla.  The  full  description  of  the  characters 
of  these  groups  will  be  more  conveniently  taken  later  (p.  12). 
In  some  cases,  especially  among  the  bacilli,  there  may  occur 
under  certain  circumstances  changes  in  the  protoplasm  whereby 
a  resting  stage  or  spore  is  formed. 

The  higher  forms  show  advance  on  the  lower  along  two  lines. 
(1)  On  the  one  hand,  they  consist  of  filaments  made  up  of 
simple  elements  such  as  occur  in  the  lower  forms.  These 
filaments  may  be  more  or  less  septate,  may  be  provided  with  a 


2-  ^:    GENERA*. ^MORPHOLOGY  AND  BIOLOGY 


>aili,.  and  ^may.-sjiojv^  branching  either  true  or  false.  The 
minute  structure  of  the  elements  comprising  these  filaments  is 
analogous  to  that  of  the  lower  forms.  Their  size,  however,  is 
often  somewhat  greater.  The  lower  forms  sometimes  occur  in 
filaments,  but  here  every  member  of  the  filament  is  independent, 
while  in  the  higher  forms  there  seems  to  be  a  certain  inter- 
dependence among  the  individual  elements.  For  instance, 
growth  may  occur  only  at  one  end  of  a  filament,  the  other 
forming  an  attachment  to  some  fixed  object.  (2)  The  higher 
forms,  moreover,  present  this  further  development,  that  in  certain 
cases  some  of  the  elements  may  be  set  apart  for  the  reproduction 
of  new  individuals. 

Terminology. — The  term  bacterium  of  course  in  strictness 
only  refers  to  the  rod-shaped  varieties  of  the  group,  but  as  it 
has  given  the  name  bacteriology  to  the  science  which  deals  with 
the  whole  group,  it  is  convenient  to  apply  it  to  all  the  members 
of  the  latter,  and  to  reserve  the  term  bacillus  for  the  rod-shaped 
varieties.  Other  general  words,  such  as  germ,  microbe,  micro- 
organism, are  used  as  synonymous  with  bacterium,  though  these 
are  often  made  to  include  the  smallest  organisms  of  the  animal 
kingdom. 

While  no  living  organisms  lower  than  the  bacteria  are  known 
(though  certain  facts  regarding  ultra-microscopic  forms  of  life 
make  the  occurrence  of  such  possible),  the  upper  limits  of  the 
group  are  difficult  to  define,  and  it  is  further  impossible  in  the 
present  state  of  our  knowledge  to  give  other  than  a  provisional 
classification  of  the  forms  which  all  recognise  to  be  bacteria. 
The  division  into  lower  and  higher  forms,  however,  is  fairly  well 
marked,  and  we  shall  therefore  refer  to  the  former  as  the  lower 
bacteria,  and  to  the  latter  as  the  higher  bacteria. 

Morphological  Relations.  —The  relations  of  the  bacteria  to  the  animal 
kingdom  on  the  one  hand  and  to  the  vegetable  on  the  other  constitute  a 
somewhat  difficult  question.  It  is  best  to  think  of  there  being  a  group 
of  small,  unicellular  organisms,  which  may  represent  the  most  primitive 
forms  of  life  before  differentiation  into  animal  and  vegetable  types  had 
occurred.  This  would  include  the  flagellata  and  infusoria,  the  myxo- 
mycetes,  the  lower  algre.  and  the  bacteria.  To  the  lower  algae  the  bacteria 
show  many  similarities.  These  algae  are  unicellular  masses  of  protoplasm, 
having  generally  the  same  shapes  as  the  bacteria,  and  largely  multiply  by 
fission.  Endogenous  sporulation,  however,  does  not  occur,  nor  is  motility 
necessarily  associated  with  the  possession  of  flagella.  Also  their  proto- 
plasm differs  from  that  of  the  bacteria  in  containing  chlorophyll  and 
another  blue-green  pigment  called  phycocyan.  From  the  morphological 
resemblances,  however,  between  these  algse  and  the  bacteria,  and  from 
the  fact  that  fission  plays  a  predominant  part  in  the  multiplication  of 
both,  they  have  been  grouped  together  in  one  class  as  the  Schizophyta 


THE  STRUCTURE  OF  THE  BACTERIAL  CELL   3 

or  splitting  plants  (German,  Spaltpflanzen).  And  of  the  two  divisions 
forming  these  Schizophyta  the  splitting  algae  are  denominated  the 
sdiizophycoe  (German,  Spaltalgen),  while  the  bacteria  or  splitting  fungi 
are  called  the  schizomycetes  (German,  Spaltpilzen).  The  bacteria  are, 
thrivf'oi-e,  oftm  spoken  of  as  the  schizomycetes.  Certain  bacteria  which 
have  been  described  as  containing  chlorophyll  ought  probably  to  be 
grouped  among  the  schizophycese. 


GENERAL  MORPHOLOGY  OF  THE  BACTERIA. 

The  Structure  of  the  Bacterial  Cell. — On  account  of  the 
minuteness  of  bacteria  the  investigation  of  their  structure  is 
attended  with  great  difficulty.  When  examined  under  the 
microscope,  in  their  natural  condition,  e.g.  in  water,  they  appear 
merely  as  colourless  refractile  bodies  of  the  different  shapes 
named.  Spore  formation  and  motility,  when  these  exist,  can 
also  be  observed,  but  little  else  can  be  made  out.  For  their 
propei-  investigation  advantage  is  always  taken  of  the  fact  of 
their  attinities  for  various  dyes,  especially  those  which  are  usually 
chosen  as  good  stains  for  the  nuclei  of  animal  cells.  Certain 
points  have  thus  been  determined.  The  bacterial  cell  consists 
of  a  sharply  contoured  mass  of  protoplasm  which  reacts  to, 
especially  basic,  aniline  dyes  like  the  nucleus  of  an  animal  cell 
— though  from  this  fact  we  cannot  deduce  that  the  two  are 
identical  in  composition.  A  healthy  bacterium  when  thus 
stained  presents  the  appearance  of  a  finely  granular  or  almost 
homogeneous  structure.  The  protoplasm  is  surrounded  by  an 
envelope  which  can  in  some  cases  be  demonstrated  by  over- 
staining  a  specimen  with  a  strong  aniline  dye,  when  it  will  appear 
s  halo  round  the  bacterium.  This  envelope  may  sometimes 
oe  seen  to  be  of  considerable  thickness.  Its  innermost  layer  is 
probably  of  a  denser  consistence,  and  sharply  contours  the 
contained  protoplasm,  giving  the  latter  the  appearance  of  being 
surrounded  by  a  membrane.  It  is  only,  however,  in  some  of 
the  higher  forms  that  a  definite  membrane  occurs.  Sometimes 
the  outer  margin  of  the  envelope  is  sharply  defined,  in  which  case 
the  l.acterinm  appears  to  have  a  distinct  capsule,  and  is  known 
as  a  capsulated  bacterium  (vide  Fig.  1,  No.  4;  and  Fig.  62). 
The  cohesion  of  bacteria  into  masses  depends  largely  on  the 
character  of  the  envelope.  If  the  latter  is  glutinous,  then  a 
l:ir^e  mass  of  the  same  species  may  occur,  formed  of  individual 
Bacteria  embedded  in  what  appears  to  be  a  mass  of  jelly.  When 
this  occurs,  it  is  known  as  a  zoogloea  mass.  On  the  other  hand, 
it  the  envelope  has  not  this  cohesive  property  the  separation  of 
individuals  may  easily  take  place,  especially  in  a  fluid  medium 


4  GENERAL  MORPHOLOGY  AND  BIOLOGY 

in  which  they  may  float  entirely  free  from  one  another.  Many 
of  the  higher  bacteria  possess  a  sheath  which  has  a  much  more 
definite  structure  than  is  found  among  the  lower  forms.  It 
resists  external  influences,  possesses  elasticity,  and  serves  to  bind 
the  elements  of  the  organism  together. 

Reproduction  among  the  Lower  Bacteria. — When  a  bacterial 
cell  is  placed  in  favourable  surroundings,  it  multiplies ;  as  has 
been  said,  this,  in  the  great  majority  of  cases,  takes  place  by 
simple  fission.  In  the  process  a  constriction  appears  in  the 
middle  and  a  transverse  unstained  line  develops  across  the 
protoplasm  at  that  point.  The  process  goes  on  till  two 
individuals  can  be  recognised,  which  may  remain  for  a  time 
attached  to  one  another,  or  become  separate,  according  to  the 
character  of  the  envelope,  as  already  explained.  In  most 
bacteria  growth  and  multiplication  go  on  with  great  rapidity. 
A  bacterium  may  reach  maturity  and  divide  in  from  twenty 
minutes  to  half  an  hour.  If  division  takes  places  only  every 
hour,  from  one  individual  after  twenty -four  hours  17,000,000 
similar  individuals  will  be  produced.  As  shown  by  the  results 
of  artificial  cultivation,  others,  such  as  the  tubercle  bacillus, 
multiply  much  more  slowly.  Sometimes  division  proceeds  so 
rapidly  that  the  young  individuals  do  not  reach  the  adult  size 
before  multiplication  again  occurs.  This  may  give  rise  to 
anomalous  appearances. 

From  investigations  by  Graham- Smith  and  others,  it  appears  that 
the  consistence  of  the  envelope  may  have  an  importance  in  modifying 
the  naked-eye  and  low-power  appearances  presented  by  bacterial  colonies 
which  constitute  a  feature  in  the  identification  of  species  (see  p.  137). 
Graham-Smith,  working  with  bacilli,  differentiates  four  groups — a  "loop- 
forming,"  in  which  the  envelope  is  so  tough  that,  after  division,  rupture 
but  rarely  occurs  (b.  anthracis) ;  a  "  folding  "  group,  in  which  the  envelope 
is  so  flexible  and  extensile  that  the  members  of  a  chain  can  be  folded  on 
one  another  as  successive  divisions  take  place  (b.  pestis)  ;  a  "snapping" 
group,  in  which  partial  rupture  of  the  envelope  occurs  on  division 
(b.  diphtherias);  and  a  "slipping"  group,  where  the  envelope  readily 
breaks,  and  successively  developed  bacilli  slip  past  each  other  (v.  cholerse). 

When  bacteria  are  placed  in  unfavourable  conditions  as 
regards  food,  etc.,  growth  and  multiplication  take  place  with 
difficulty.  In  the  great  majority  of  cases  this  is  evidenced  by 
changes  in  the  appearance  of  the  protoplasm.  Instead  of  its 
maintaining  the  regularity  of  shape  seen  in  healthy  bacteria, 
various  aberrant  appearances  are  presented.  This  occurs  especially 
in  the  rod-shaped  varieties,  where  flask-shaped  or  dumb-bell - 
shaped  individuals  may  be  seen.  The  regularity  in  structure 


SPORE  FORMATION  5 

ami  size  is  quite  lost.  The  appearance  of  the  protoplasm 
also  is  often  altered.  Instead  of,  as  formerly,  staining  well,  it 
does  not  stain  readily,  and  may  have  a  uniformly  pale  homo- 
geneous appearance,  while  in  an  old  culture  only  a  small 
proportion  of  the  bacteria  may  stain  at  all.  Sometimes,  on  the 
other  hand,  a  degenerated  bacterium  contains  intensely  stained 
irrannlt's  or  globules  which  may  be  of  large  size.  Such  aberrant 
;ni«l  degenerate  appearances  are  referred  to  as  involution  forms. 
That  these  forms  really  betoken  degenerative  changes  is  shown 
by  the  fact  that,  on  their  being  again  transferred  to  favourable 
conditions,  only  slight  growth  at  first  takes  place.  Many 
individuals  have  undoubtedly  died,  and  the  remainder  which 
live  and  develop  into  typical  forms  may  sometimes  have  lost 
some  of  their  properties. 

Reproduction  among  the  Higher  Bacteria. —Most  of  the  higher  bacteria 
consist  of  thread-like  structures  more  or  less  septate  and  often  surrounded 
by  a  sheath.  The  organism  is  frequently  attached  at  one  end  to  some 
object  or  to  another  individual.  It  grows  to  a  certain  length  and  then 
at  tin'  lice  end  certain  cells,  called  gonidia,  are  cast  oil'  from  which  new 
individuals  are  formed.  These  gonidia  may  be  formed  by  a  division 
taking  place  in  the  terminal  element  of  the  filament  such  as  has  occurred 
in  the  growth  of  the  latter.  In  some  cases,  however,  division  takes 
j'lai •(•  in  three  dimensions  of  space.  The  gonidia  have  a  free  existence 
fur  a  certain  time  before  becoming  attached,  and  in  this  stage  are 
sometimes  motile.  They  are  usually  rod-like  in  shape,  sometimes 
pyriform.  They  do  not  possess  any  special  powers  of  resistance. 

Spore  Forma,tion. — In  certain  species  of  the  lower  bacteria, 
under  certain  circumstances,  changes  take  place  in  the  protoplasm 
which  result  in  the  formation  of  bodies  called  spores,  to  which 
the  vital  activities  of  the  original  bacteria  are  transferred. 
Spore  formation  occurs  chiefly  among  the  bacilli  and  in  some 
spirilla.  Its  commencement  in  a  bacterium  is  indicated  by  the 
appearance  in  the  protoplasm  of  a  minute  highly  refractile 
granule  unstained  by  the  ordinary  methods.  This  increases  in 
-i/i-,  and  assumes  a  round,  oval,  or  short  rod-shaped  form,  always 
shorter  but  often  broader  than  the  original  bacterium.  In  the 
process  of  spore  formation  the  rest  of  the  bacterial  protoplasm 
may  remain  unchanged  in  appearance  and  staining  power  for  a 
considerable  time  (e.g.  b.  tetani),  or,  on  the  other  hand,  it  may 
soon  lose  its  power  of  staining  and  ultimately  disappear,  leaving 
the  spoiv  in  the  remains  of  the  envelope  (?.</.  b.  anthracis). 
This  method  of  spore  formation  is  called  cn<l<><i<  nuns.  Bacterial 
spores  are  always  non-motile.  The  spore  may  appear  in  the 
centre  of  the  bacterium,  or  it  may  be  at  one  extremity,  or  a 
short  distance  from  one  extremity  (Fig.  1,  No.  11).  In  structure 


6  GENERAL  MORPHOLOGY  AND  BIOLOGY 

the  spore  consists  of  a  mass  of  protoplasm  surrounded  by  a  dense 
membrane.  This  can  be  demonstrated  by  methods  which  will 
be  described,  the  underlying  principle  of  which  is  the  prolonged 
application  of  a  powerful  stain.  The  membrane  is  supposed  to 
confer  on  the  spore  its  characteristic  feature,  namely,  great 
capacity  of  resistance  to  external  influences  such  as  heat  or 
noxious  chemicals.  Koch,  for  instance,  in  one  series  of  experi- 
ments, found  that  while  the  bacillus  anthracis  in  the  unspored 
form  was  killed  by  a  two  minutes'  exposure  to  1  per  cent  carbolic 
acid,  spores  of  the  same  organism  resisted  an  exposure  of  from 
one  to  fifteen  days. 

When  a  spore  is  placed  in  suitable  surroundings  for  growth, 
it  again  assumes  the  original  bacillary  or  spiral  form.  The 
capsule  dehisces  either  longitudinally,  or  terminally,  or  trans- 
versely. In  the  last  case  the  dehiscence  may  be  partial,  and  the 
new  individual  may  remain  for  a  time  attached  by  its  ends  to 
the  hinged  spore-case,  or  the  dehiscence  may  be  complete  and 
the  bacillus  grow  with  a  cap  at  each  end  consisting  of  half  the 
spore-case.  Sometimes  the  spore-case  does  not  dehisce,  but  is 
simply  absorbed  by  the  developing  bacterium. 

It  is  important  to  note  that  in  the  bacteria  spore  formation 
is  rarely,  if  ever,  to  be  considered  as  a  method  of  multiplication. 
In  at  least  the  great  majority  of  cases  only  one  spore  is  formed 
from  one  bacterium,  and  only  one  bacterium  in  the  first  instance 
from  one  spore.  Sporulation  is  to  be  looked  upon  as  ^jresting 
sJ«|M_jQf--^Jba£leriuin,  and  is  to  be  contrasted  with  the  stage 
when  active  multiplication  takes  place.  The  latter  is  usually 
referred  to  as  the  vegetative .  stage^  of_lh&_bacterium.  Regarding 
the  signification  of  spore  formation  in  bacteria,  there  has  been 
some  difference  of  opinion.  According  to  one  view,  it  may  be 
regarded  as  representing  the  highest  stage  in  the  vital  activity 
of  a  bacterium.  There  is  thus  an  alternation  between  the 
vegetative  and  spore  stage,  the  occurrence  of  the  latter  being 
necessary  to  the  maintenance  of  the  species  in  its  greatest 
vitality.  Such  a  rejuvenescence,  as  it  were,  through  sporulation, 
is  known  in  many  algae.  In  support  of  this  view  there  are 
certain  facts.  In  many  cases,  for  instance,  spore  formation  only 
occurs  at  temperatures  specially  favourable  for  growth  and 
multiplication.  There  is  often  a  temperature  below  which, 
while  vegetative  growth  still  takes  place,  sporulation  will  not 
occur  ;  and  in  the  case  of  b.  anthracis,  if  the  organism  be  kept 
at  a  temperature  above  the  limit  at  which  it  grows  best,  not 
only  are  no  spores  formed,  but  the  strain  may  lose  the  power 
of  sporulation.  Furthermore,  in  the  case  of  bacteria  preferring 


SPORE  FORMATION  7 

the  presence  of  oxygen  for  their  growth,  an  abundant  supply 
of  this  gas  ma^r  favour  sporulation.  It  is  probable  that  even 
among  bacteria  preferring  the  absence  of  oxygen  for  vegetative 
uiowth,  the  presence  of  this  gas  favours  sporulation.  Some 
facts  relating  to  tlwi.1.  cases  in  which  two  spores  are  formed  in 
one  bacterium  have  been  adduced  to  support  the  view  that 
sporulation  may  represent  a  degenerate  sexual  process.  Here  a 
partial  fission  of  a  cell  has  been  observed  followed  by  a  re- 
fusion  of  the  protoplasmic  moieties  and  the  formation  of  a  spore 
at  each  end  of  the  rod.  The  second  view  with  regard  to 
sporulation  is  that  a  bacterium  only  forms  a  spore  when  its 
surroundings,  especially  its  food  supply,  become  unfavourable 
for  vegetative  growth ;  it  then  remains  in  this  condition  until  it 
is  placed  in  more  suitable  surroundings.  Such  an  occurrence 
would  be  analogous  to  what  takes  place  under  similar  conditions 
in  many  of  the  protozoa.  Often  sporulation  can  be  prevented 
from  taking  place  for  an  indefinite  time  if  a  bacterium  is 
constantly  supplied  with  fresh  food  (the  other  conditions  of  life 
being  equal).  The  presence  of  substances  excreted  by  the 
bacteria  themselves  plays,  however,  a  more  important  part  in 
making  the  surroundings  unfavourable  than  the  mere  exhaustion 
of  the  food  supply.  A  living  spore  will  always  develop  into  a 
vegetative  form  if  placed  in  a  fresh  food  supply.  With  regard  to 
the  rapid  formation  of  spores  when  the  conditions  are  favourable 
for  vegetative  growth,  it  must  be  borne  in  mind  that  in  such 
circumstances  the  conditions  may  really  very  quickly  become 
unfavourable  for  a  continuance  of  growth,  since  not  only  will  the 
food  supply  around  the  growing  bacteria  be  rapidly  exhausted, 
but  the  excretion  of  effete  and  inimical  matters  will  be  all  the 
more  rapid. 

\\V  must  note  that  the  usually  applied  tests  of  a  body 
developed  within  a  bacterium  being  a  spore  are  (1)  its  staining 
reaction,  namely,  resistance  to  ordinary  staining  fluids,  but 
capacity  of  being  stained  by  the  special  methods  devised  for 
the  purpose  (vide  p.  109) ;  (2)  the  fact  that  the  bacterium 
containing  the  spore  has  higher  .powers  of  resistance  against 
inimical  conditions  than  a  vegetative  form.  It  is  important  to 
bear  these  tests  in  mind,  as,  in  some  of  the  smaller  bacteria 
especially,  it  is  very  difficult  to  say  whether  they  spore  or  not. 
There  may  appear  in  such  organisms  small  unstained  spots,  the 
significance  of  which  it  is  very  difficult  to  determine. 

The  Question  of  Arthrosporous  Bacteria.— It  is  stated  by  Hueppe  that 
aiming  certain  organi.Nins,  <\<j.  some  streptococci,  certain  individuals  may, 
without  endogenous  sporulation,  take  on  a  resting  stage.  These  become 


8  GENERAL  MORPHOLOGY  AND  BIOLOGY 

swollen,  stain  well  with  ordinary  stains,  and  they  are  stated  to  have 
higher  power  of  resistance  than  the  other  forms  ;  further,  when  vegetative 
life  again  occurs,  it  is  from  them  that  multiplication  is  said  to  take  place. 
From  the  fact  that  there  is  no  new  formation  within  the  protoplasm, 
but  that  it  is  the  whole  of  the  latter  which  participates  in  the  change, 
these  individuals  have  been  called  arthrospores.  The  existence  of  such 
special  individuals  amongst  the  lower  bacteria  is  extremely  problematical. 
They  have  no  distinct  capsule,  and  they  present  no  special  staining 
reactions,  nor  any  microscopic  features  by  which  they  can  be  certainly 
recognised,  while  their  alleged  increased  powers  of  resistance  are  very 
doubtful.  All  the  phenomena  noted  can  be  explained  by  the  undoubted 
fact  that  in  an  ordinary  growth  there  is  very  great  variation  among 
the  individual  organisms  in  their  powers  of  resistance  to  external 
conditions. 

Motility. — As  has  been  stated,  many  bacteria  are  motile. 
Motility  can  be  studied  by  means  of  hanging-drop  preparations 
(vide  p.  69).  The  movements  are  of  a  darting,  rolling,  or 
vibratile  character.  The  degree  of  motility  depends  on  the 
species,  the  temperature,  the  age  of  the  growth,  and  on  the 
medium  in  which  the  bacteria  are  growing.  Sometimes  the 
movements  are  most  active  just  after  the  cell  has  multiplied, 
sometimes  it  goes  on  all  through  the  life  of  the  bacterium, 
sometimes  it  ceases  when  sporulation  is  about  to  occur.  Motility 
is  associated  with  the  possession  of  fine  wavy  thread-like 
appendages  called  flagella,  which  for  their  demonstration  require 
the  application  of  special  staining  methods  (vide  Fig.  1,  No.  12-; 
and  Fig.  108).  They  have  been  shown  to  occur  in  many  bacilli 
and  spirilla,  but  only  in  a  few  species  of  cocci.  They  vary  in 
length,  but  may  be  several  times  the  length  of  the  bacterium, 
and  may  be  at  one  or  both  extremities  or  all  round.  When 
terminal  they  may  occur  singly  or  there  may  be  several.  The 
nature  of  these  fiageHa  has  been  much  disputed.  Some  have 
held  that,  unlike  what  occurs  in  many  algae,  they  are  not  actual 
prolongations  of  the  bacterial  protoplasm,  but  merely  appendages 
of  the  envelope,  and  have  doubted  whether  they  are  really  organs 
of  locomotion.  There  is  now,  however,  little  doubt  that  they 
belong  to  the  protoplasm.  By  appropriate  means  the  central 
parts  of  the  latter  can  be  made  to  shrink  away  from  the  peripheral 
(vide  infra,  "  plasmolysis  ").  In  such  a  case  movement  goes  on 
as  before,  and  in  stained'  preparations  the  flagella  can  be  seen 
to  be  attached  to  the  peripheral  zone.  It  is  to  be  noted  that 
flagella  have  never  been  demonstrated  in  non-motile  bacteria, 
while,  on  the  other  hand,  they  have  been  observed  in  nearly  all 
motile  forms.  There  is  little  doubt,  however,  that  all  cases  of 
motility  among  the  bacteria  are  not  dependent  on  the  possession 
of  flagella,  for  in  some  of  the  special  spiral  forms,  and  in  most 


STRUCTURE  OF  BACTERIAL  PROTOPLASM         9 

of  the  higher  bacteria,  motility  is  probably  due  to  contractility 
of  the  protoplasm  itself. 

The  Minuter  Structure  of  the  Bacterial  Protoplasm. — Many  attempts 
have  been  made  to  obtain  deeper  information  as  to  the  structure  of  the 
bacterial  cell,  and  especially  as  to  its  behaviour  in  division.  These 
ha vi-  largely  turned  on  the  interpretation  to  1m  put  on  certain  appear- 
ances which  have  been  observed.  These  appearances  are  of  two  kinds. 
First,  under  certain  circumstances  irregular  deeply-stained  grannies  are 
•  •liM-m-d  in  the  protoplasm,  often,  when  they  occur  in  a  bacillus,  giving 
the  latter  the  appearance  of  a  short  chain  of  cocci.  They  are  often 
called  metachromatic  granules  (vide  Fig.  1,  No.  16)  from  the  fact  that 
by  appropriate  procedure  they  can  be  stained  with  one  dye,  and  the 
protoplasm  in  which  they  lie  with  another  ;  sometimes,  when  a  single 
stain  is  used,  such  as  methylene  blue,  they  assume  a  slightly  different 
tint  from  the  protoplasm. 

For  the  demonstration  of  the  metachromatic  granules  two  methods 
have  been  advanced.  Ernst  recommends  that  a  few  drops  of  Lofflers 
methylene  blue  (vide  p.  104)  be  placed  on  a  cover-glass  preparation  and 
the  latter  passed  backwards  and  forwards  over  a  Bunsen  flame  for  half 
a  minute  after  steam  begins  to  rise.  The  preparation  is  then  washed 
in  water  and  counter-stained  for  one  to  two  minutes  in  watery  Bismarck- 
brown.  The  granules  are  here  stained  blue,  the  protoplasm  brown. 
Neisser  stains  a  similar  preparation  in  warm  carbol-fuchsin,  washes 
with  1  per  cent,  sulphuric  acid,  and  counter-stains  with  Loffler's  blue. 
Here  the  granules  are  magenta,  the  protoplasm  blue.  The  general 
character  of  the  granules  thus  is  that  they  retain  the  first  stain  more 
intensely  than  the  rest  of  the  protoplasm  does. 

A  second  appearance  which  can  sometimes  be  seen  in  specimens 
stained  in  ordinary  ways  is  the  occurrence  of  a  concentration  of  the 

Protoplasm  at  each  end  of  a  bacterium,  indicated  by  these  parts  being 
eeply  stained.     These  deeply  stained  parts  are  sometimes  called  polar 
granules  (vide  Fig.  1,  No.  16,  the  bacillus  most  to  the  right)  (German, 
Polkiirnchen  or  Polkorner). 

With  regard  to  the  significance  that  is  to  be  attached  to  such 
appearances,  much  depends  on  whether  they  are  constantly  present 
under  all  circumstances,  or  only  occasionally,  when  the  organism  is 
grown  in  special  media  or  under  special  growth  conditions.  Some 
bacteria,  however  stained,  show  evidence  of  having  the  protoplasm 
somewhat  granular,  e.g.  the  diphtheria  bacillus.  In  other  cases  this 
granular  condition  is  only  seen  when  the  organism  has  been  grown  under 
bad  conditions,  or  where  the  food  supply  is  becoming  exhausted.  Some 
have  thought  that  the  appearances  might  be  due  to  a  process  allied  to 
mitosis  and  might  signify  approaching  division,  but  of  this  there  is  no 
evidence. 

In  perfect  healthy  and  young  bacteria,  appearances  of  granule 
formation  and  of  vacuolation  may  be  accidentally  produced  by  physical 
means  in  the  occurrence  of  what  is  known  as  pltumotyffa  To  speak 
generally,  when  a  mass  of  protoplasm  surrounded  by  a  fairly  linn 
envelope  of  a  colloidal  nature  is  placed  in  a  solution  containing  salts  in 
greater  concentration  than  that  in  which  it  has  previously  been  living, 
then  by  a  process  of  osmosis  the  water  held  in  the  protoplasm  passes 
out  through  the  membrane,  and,  the  protoplasm  retracting  from  the 
latt- T,  the  appearance  of  vacuolation  is  presented.  Now,  in  making  a 


10         GENERAL  MORPHOLOGY  AND  BIOLOGY 

dried  film  for  the  microscopic  examination  of  bacteria,  the  conditions 
necessary  for  the  occurrence  of  this  process  may  be  produced,  and  the 
appearances  of  vacuolation  and,  in  certain  cases,  of  Polkorner  may  thus 
be  brought  about.  Plasmolysis  in  bacteria  has  been  extensively 
investigated,1  and  has  been  found  to  occur  in  some  species  more  readily 
than  in  others.  Furthermore,  it  is  often  most  readily  observed  in  old  or 
otherwise  enfeebled  cultures. 

Biitschli,  from  a  study  of  some  large  sulphur-containing  forms,  con- 
cludes that  the  greater  part  of  the  bacterial  cell  may  correspond  to  a 
nucleus,  and  that  this  is  surrounded  by  a  thin  layer  of  protoplasm  which 
in  the  smaller  bacteria  escapes  notice,  unless  when,  as  in  the  bacilli,  it 
can  be  made  out  at  the  ends  of  the  cells.  Fischer,  it  may  be  said,  looks 
on  the  appearances  seen  in  Biitschli's  preparations  as  due  to  plasmolysis. 

The  Chemical  Composition  of  Bacteria. — In  the  bodies  of 
bacteria  many  definite  substances  occur.  Some  bacteria  have 
been  described  as  containing  chlorophyll,  but  these  are  properly 
to  be  classed  with  the  schizophyceae.  Sulphur  is  found  in  some 
of  the  higher  forms,  and  starch_gramiles  areTalso  described  as 
occurring.  Many  species  of  bacteria,  when  growing  in  masses, 
are  brilliantly  coloured,  though  few  bacteria  associated  with  the 
production  of  disease  give  rise  to  pigments.  In  some  of  the 
organisms  classed  as  bacteria  a  pigment  named  bacterio-purpurin 
has  been  observed  in  the  protoplasm,  and  similar  intracellular 
pigments  probably  occur  in  some  of  the  larger  forms  of  the 
lower  bacteria  and  may  occur  in  the  smaller  ;(but  it  is  usually 
impossible  to  determine  whether  the  pigment  occurs  inside  or 
outside  the  protoplasm/)  In  many  cases,  for  the  free  production 
of  pigment  abundant  oxygen  supply  is  necessary  ;  but  sometimes, 
as  in  the  case  of  spirillum  rubrum,  the  pigment  is  best  formed 
in  the  absence  of  oxygen.  Sometimes  the  faculty  of  forming  it 
may  be  lost  by  an  organism  for  a  time,  if  not  permanently,  by 
the  conditions  of  its  growth  being  altered.  Thus,  for  example, 
if  the  b.  pyocyaneus  be  exposed  to  the  temperature  of  42°  C. 
for  a  certain  time,  it  loses  its  power  of  producing  its  bluish 
pigment.  Pigments  formed  by  bacteria  often  diffuse  out  into, 
and  colour,  the  medium  for  a  considerable  distance  around. 

Comparatively  little  is  known  of  the  nature  of  bacterial  pigments. 
Zopf,  however,  has  found  that  many  of  them  belong  to  a  group  of 
colouring  matters  which  occur  widely  in  the  vegetable  and  animal 
kingdoms,  namely,  the  lipochromes.  These  lipochromes,  which  get  their 
name  from  the  colouring  matter  of  animal  fat,  include  the  colouring 
matter  in  the  petals  of  Ranunculacese,  the  yellow  pigments  of  serum  and 
of  the  yolks  of  eggs,  and  many  bacterial  pigments.  The  lipochromes  are 
characterised  by  their  solubility  in  chlorolorm,  alcohol,  ether,  and 

1  Consult  Fischer,  "  Untersuchungen  iiber  Bakterien,"  Berlin,  1894; 
"  Ueber  den  Bau  der  Cyauophyceen  mid  Bakterieu,"  Jena,  1897. 


THE  CLASSIFICATION  OF  BACTERIA  11 

petroleum,  and  by  their  giving  indigo-blue  crystals  with  strong  sulphuric 
arid,  and  a  green  colour  with  iodine  dissolved  in  potassium  iodide. 
Though  cry stal line  compounds  of  these  have  been  obtained,  their 
cht  inical  constitution  is  entirely  unknown,  and  even  their  percentage 
i  (imposition  is  disputed. 

Some  observations  have  been  made  on  the  chemical  structure 
of  bacterial  protoplasm.  Nencki  isolated  from  the  bodies  of 
certain  putrefactive  bacteria  proteid  bodies  which,  according  to 
Kuppel,  appear  to  have  been  allied  to  peptone,  and  which 
differed  from  nucleo-proteids  in  not  containing  phosphorus, 
but  many  of  the  proteids  isolated  by  other  chemists  have 
been  allied  in  their  nature  to  the  protoplasm  of  the  nuclei 
of  cells.  Buchner  in  certain  researches  obtained  bodies  of  this 
nature  allied  to  the  vegetable  caseins,  and  he  adduces  evidence 
to  show  that  it  is  to  these  that  the  characteristic  staining 
properties  are  due.  Various  observers  have  isolated  similar 
phosphorus-containing  proteids  from  different  bacteria.  Besides 
proteids,  however,  substances  of  a  different  nature  have  been 
isolated.  Thus  cellulose,  fatty  material,  chitin,  wax-like  bodies, 
and  other  substances  have  been  observed.  There  are  also  found 
various  mineral  salts,  especially  those  of  sodium,  potassium,  and 
magnesium.  The  amount  of  different  constituents  varies  ac- 
cording to  the  age  of  the  culture  and  the  medium  used  for 
m<>\\tli,  and  certainly  great  variation  takes  place  in  the  com- 
position of  different  species. 

The  Classification  of  Bacteria. — There  have  been  numerous 
schemes  set  forth  for  the  classification  of  bacteria,  the  fuixla- 
nii'iital  principle  running  through  all  of  which  has  been  the 
recognition  of  the  two  sub-groups  and  the  type  forms  mentioned 
in  the  opening  paragraph  above.  In  the  attempts  to  still 
furl  her  subdivide  the  group,  scarcely  two  systematists  are  agreed 
a-  to  the  characters  on  which  sub-classes  are  to  be  based.  Our 
(•resent  knowledge  of  the  essential  morphology  and  relations  of 
bacteria  is  as  yet  too  limited  for  a  really  natural  classification 
to  be  attempted.  To  prepare  for  the  elaboration  of  the  latter, 
Marshall  Ward  suggested  that  in  every  species  there  should  be 
studied  the  habitat,  best  food  supply,  condition  as  to  gaseous 
environment,  range  of  growth  temperature,  morphology,  life 
hUtoiy.  >pecial  properties,  and  pathogen icity.  Some  recent 
at!  i 'in  j.ts  to  carry  out  such  a  plan  will  be  referred  to  in 
•  -onnectioii  with  the  principles  of  general  bacteriological 
diagnosis  (p.  135). 

\\V  must  thus  be,  content  with  a  provisional  and  incomplete 
We  have  said  that  the  division  into  lower  and 


12         GENERAL  MORPHOLOGY  AND  BIOLOGY 

higher  bacteria  is  recognised  by  all,  though,  as  in  every  other 
classification,  transitional  forms  have  to  be  accounted  for.  In 
subdividing  the  bacteria  further,  the  forms  they  assume  con- 
stitute at  present  the  only  practicable  basis  of  classification. 
The  lower  bacteria  thus  naturally  fall  into  the  three  groups 
mentioned,  the  cocci,  bacilli,  and  spirilla,  though  the  higher 
are  more  difficult  to  deal  with.  Subsidiary,  though  important, 
points  in  still  further  subdivision  are  the  planes  in  which  fission 
takes  place  and  the  presence  or  absence  of  spores.  The  recogni- 
tion of  actual  species  is  often  a  matter  of  great  difficulty.  The 
points  to  be  observed  in  this  will  be  discussed  later  (p.  137). 

I.  The  Lower  Bacteria.1 — These,  as  we  have  seen,  are 
minute  unicellular  masses  of  protoplasm  surrounded  by  an 
envelope,  the  total  vital  capacities  of  a  species  being  represented 
in  every  cell.  They  present  three  distinct  type  forms,  the 
coccus,  the  bacillus,  and  the  spirillum ;  endogenous  sporulation 
may  occur.  They  may  also  be  motile. 

1.  The  Cocci. — In  this  group  the  cells  range  in  different 
species  from  '5  /u,  to  2  /x,  in  diameter,  but  most  measure  about  1  //. 
Before  division  they  may  increase  in  size  in  all  directions.  The 
species  are  usually  classified  according  to  the  method  of  division. 
If  the  cells  divide  only  in  one  axis,  and  through  the  consistency 
of  their  envelopes  remain  attached,  then  a  chain  of  cocci  will  be 
formed.  A  species  in  which  this  occurs  is  known  as  a  strepto- 
coccus. If  division  takes  place  irregularly,  the  resultant  mass  may 
be  compared  to  a  bunch  of  grapes,  and  the  species  is  often  called 
a  staphylococcus.  Division  may  take  place  in  two  axes  at  right 
angles  to  one  another,  in  which  case  cocci  adherent  to  each  other 
in  packets  of  four  (called  tetrads)  or  sixteen  may  be  found, 
the  former  number  being  the  more  frequent.  To  all  these  forms 
the  word  micrococcus  is  often  generally  applied.  The  individuals 
in  a  growth  of  micrococci  often  show  a  tendency  to  remain 
united  in  twos.  These  are  spoken  of  as  diplococci,  but  this  is 
not  a  distinctive  character,  since  every  coccus  as  a  result  of 
division  becomes  a  diplococcus,  though  in  some  species  the 
tendency  to  remain  in  pairs  is  well  marked.  The  adhesion  of 
cocci  to  one  another  depends  on  the  character  of  the  capsule. 
Often  this  has  a  well-marked  outer  limit  (micrococcus  tetrayenus), 
sometimes  it  is  of  great  extent,  its  diameter  being  many  times 
that  of  the  coccus  (streptococcus  mcsenteriodes).  It  is  especially 
among  the  streptococci  and  staphylococci  that  the  phenomenon 
of  the  formation  of  arthrospores  is  said  to  occur.  In  none  of 

1  For  the  illustration   of  this  and  the  succeeding  systematic  paragraphs, 
vide  Fig.  1. 


THE  LOWER  BACTERIA 


13 


156 


Fio.  1.— 1.  Coccus.  2.  Streptococcus.  3.  Staphylococcus.  4.  Capsulated  diplococcus. 
5.  "  Biscuit  "-shaped  coccua.  6.  Tetrads.  7.  Sarcina  fonn.  8.  Types  of  bacilli 
(1-8  are  diagrammatic).  9.  Non-septate  spirillum  x  1000.  10.  Ordinary  spirillum — 
(a)  comma-shaped  element ;  (t>)  formation  of  spiral  by  comma-shaped  elements 
x  1000.  11.  Types  of  spore  formation.  12.  Flagellated  bacteria.  13.  Changes  in 
I.;K trria  produced  by  plasmolysis  (after  Fischer).  14.  Bacilli  with  terminal  proto- 
plasm (liutschli).  15.  (a)  Bacillus  composed  of  five  protoplasmic  meshes;  (6)proto- 
plMmio network  in  micrococcus  (Biitschli).  10.  Bacteria  containing  metachromatic 
granules  (Ernst,  Neisser)— some  contain  polar  granules.  17.  Beggiatoaalba.  Both 
filaments  contain  sulphur  granules — one  is  septate.  18.  Thiothrix  tennis  (\Vino- 
ki).  19.  Leptothrix  innominate  (Miller).  20.  Cladothrix  dichotoma  (Zopf). 
•.M.  Stn-jttuthrix  a<-tiiii)mvces(Bostr6m),  (a)  colony  under  low  power;  (b)  filament 
>li(.\vin«;  true  branching  ;' («)  filament  containing  coccus-like  bodies;  (d)  filament 
with  chili  at  end. 


14         GENERAL  MORPHOLOGY  AND  BIOLOGY 

the  cocci  have  endogenous  spores  been  certainly  observed.  The 
species  of  the  streptococci  and  staphylococci  differentiated 
number  several  hundreds.  Usually  included  in  this  group  are 
coccus-like  organisms  which  divide  in  three  axes  at  right  angles 
to  one  another.  These  are  usually  referred  to  as  sarcince.  If 
the  cells  are  lying  single  they  are  round,  but  usually  they  are 
seen  in  cubes  of  eight  with  the  sides  which  are  in  contact 
slightly  flattened.  Large  numbers  of  such  cubes  may  be  lying 
together.  The  sarcinse  are,  as  a  rule,  rather  larger  than  the 
other  members  of  the  group.  Most  of  the  cocci  are  non-motile, 
but  a  few  motile  species  possessing  flagella  have  been  described. 

2.  Bacilli. — These  consist  of  long  or  short  cylindrical  cells, 
with  rounded  or  sharply  rectangular  ends,  usually  not  more  than 
1  [A  broad,  but  varying  very  greatly  in  length.     They  may  be 
motile    or   non-motile.       Where    flagella    occur,    these    may   be 
distributed  all  round  the  organism,  or  only  at  one  or  both  of 
the  poles   (pseudomonas).     Several   species    are    provided    with 
sharply-marked    capsules    (b.    pneumonias).       In   many   species 
endogenous  sporulation  occurs.     The  spores  may  be  central  or 
terminal,  round,  oval,  or  spindle-shaped. 

Great  confusion  in  nomenclature  has  arisen  in  this  group  in  con- 
sequence of  the  different  artificial  meanings  assigned  to  the  essentially 
synonymous  terms  bacterium  and  bacillus.  Migula,  for  instance,  applies 
the  former  term  to  non-motile  species,  the  latter  to  the  motile.  Hueppe, 
on  the  other  hand,  calls  those  in  which  endogenous  sporulation  does 
not  occur,  bacteria,  and  those  where  it  does,  bacilli.  In  the  ordinary 
terminology  of  systematic  bacteriology  the  word  bacterium  has  been 
almost  dropped,  and  is  reserved,  as  we  have  done,  as  a  general  term  for 
the  whole  group.  It  is  usual  to  call  all  the  rod-shaped  varieties  bacilli. 

3.  Spirilla. — These  consist  of  cylindrical  cells  more  or  less 
spiral  or  wavy.     Of  such  there  are  two  main  types.     In  one  there 
is  a  long  non-septate,   usually  slender,   wavy  or  spiral  thread 
(Fig.  1,  No.  9).     In  the  other  type  the  unit  is  a  short  curved 
rod   (often  referred  to  as  of   a  "comma"   shape).     When  two 
or  more  of  the    latter   occur,    as   they    often   do,    end   to    end 
with  their  curves  alternating,  then    a   wavy    or  spiral    thread 
results.     An  example  of  this  is  the  cholera  microbe   (Fig.    1, 
No.  10).     This  latter  type  is  of  much  more  frequent  occurrence, 
Among  the  first  group  motility  is  often  not  associated,  as  far 
as  is  known,  with  the  possession   of  flagella.      The  cells  here 
apparently  move  by  an  undulating  or  screw-like  contraction  of 
the  protoplasm.     Most  of  the  motile  spirilla,  however,  possess 
flagella.     Of  the  latter  there  may  be  one  or  two,  or  a  bunch 
containing  as  many  as  twenty,  at  one  or  both  poles.     Division 


THE  HIGHER  BACTERIA  15 

takes  place  as  among  the  bacilli,  but  in  some  of  the  non- 
-cptate  forms  a  longitudinal  fission  may  occur.  In  some  species 
endogenous  sporulation  has  been  observed. 

Three  terras  are  used  in  dividing  this  group,  to  which  different  authors 
have  given  different  meanings.  These  terms  are  spirillum,  spirochaete, 
vibrio.  Migula  makes  "  vibrio"  synonymous  with  "  microspira,"  which 
la-  applies  to  members  of  the  group  which  possess  only  one  or  two  polar 
fla^clla  ;  "spirillum "  he  applies  to  similar  species  which  have  bunches 
of  polar  Hagella,  while  "  spirochsete  "  is  reserved  for  the  long  unHagellated 
spiral  cells.  Hueppe  applies  the  term  "  spirochsete "  to  forms  without 
endospores,  "  vibrio  "  to  those  with  endospores  in  which  during  sporula- 
tion the  organism  changes  its  form,  and  "spirillum"  to  the  latter 
when  no  change  of  form  takes  place  in  sporulation.  Flugge,  another 
systematist,  applies  " spirochsete  "  ancl  "spirillum"  indiscriminately  to 
any  wavy  or  corkscrew  form,  and  "vibrio"  to  forms  where  the  undula- 
tions are  not  so  well  marked.  It  is  thus  necessary,  in  denominating  such 
a  bacterium  by  a  specific  name,  to  give  the  authority  from  whom  the 

name  is  taken. 

« 

Within  recent  years  great  doubt  has  arisen  as  to  whether  many 
of  the  non-septate  spirillary  forms,  e.g.  Spirochcete  pallida,  are 
to  be  looked  on  as  bacteria  at  all, — the  view  being  taken  that 
in,  it  may  be,  many  cases  they  represent  a  stage  in  the  life 
history  of  what  are  really  protozoa.  The  ultimate  classification 
of  this  group  of  bacteria  must  thus  be  left  an  open  question, 
and  at  present  it  is  convenient  to  denominate  the  non-septate 
spiral  rods  Spirochcete,  and  those  whose  vital  unit  is  a  single 
curved  rod  Spirilla, 

II.  The  Higher  Bacteria. — These  show  advance  on  the  lower 
in  consisting  of  definite  filaments  branched  or  unbranched.  In 
most  cases  the  filaments  at  more  or  less  regular  intervals  are 
cut  by  septa  into  short  rod-shaped  or  curved  elements.  Such 
elements  are  more  or  less  interdependent  on  one  another,  and 
special  staining  methods  are  often  necessary  to  demonstrate  the 
septa  which  demarcate  the  individuals  of  a  filament.  There  is 
further  often  a  definite  membrane  or  sheath  common  to  all  the 
elements  in  a  filament.  Not  only,  however,  is  there  this  close 
organic  relationship  between  the  elements  of  the  higher  bacteria, 
but  there  is  also  interdependence  of  function ;  for  example,  one 
end  of  a  filament  is  frequently  concerned  merely  in  attaching 
the  organism  to  some  other  object.  The  greatest  advance,  how- 
ever, consists  in  the  setting  apart  among  most  of  the  higher 
bacteria  of  the  free  terminations  of  the  filaments  for  the  produc- 
tion of  new  individuals,  as  has  been  described  (p.  5).  There 
are  various  classes  under  which  the  species  of  the  higher  bacteria 
are  grouped;  but  our  knowledge  of  them  is  still  somewhat 


16         GENERAL  MORPHOLOGY  AND  BIOLOGY 

limited,  as  many  of  the  members  have  not  yet  been  artificially 
cultivated.  The  beggiatoa  group  consists  of  free  swimming 
forms,  motile  by  undulating  contractions  of  their  protoplasm. 
For  the  demonstration  of  the  rod-like  elements  of  the  filaments 
special  staining  is  necessary.  The  filaments  have  no  special 
sheath,  and  the  protoplasm  contains  sulphur  granules.  The 
method  of  reproduction  is  doubtful.  The  thiothrix  group  re- 
sembles the  last  in  structure,  and  the  protoplasm  also  contains 
sulphur  granules;  but  the  filaments  are  attached  at  one  end, 
and  at  the  other  form  gonidia.  A  leptothrix  group  is  usually 
described  which  closely  resembles  the  thiothrix  group,  except  that 
the  protoplasm  does  not  contain  sulphur  granules.  It  cannot, 
however,  be  with  certainty  said  whether  such  organisms  can  be 
sufficiently  differentiated  from  the  bacilli  to  warrant  their  being 
placed  among  the  higher  bacteria.  In  the  cladothrix  group 
there  is  the  appearance  of  branching,  which,  however,  is  of  a 
false  kind.  Whaf  happens  is  that  a  terminal  cell  divides,  and 
on  dividing  again,  it  pushes  the  product  of  its  first  division  to 
one  side.  There  are  thus  two  terminal  cells  lying  side  by  side, 
and  as  each  goes  on  dividing,  the  appearance  of  branching  is 
given.  Here,  again,  there  is  gonidium  formation;  and  while 
the  parent  organism  is  in  some  of  its  elements  motile,  the  gonidia 
move  by  means  of  flagella.  The  highest  development  is  in  the 
streptothrix  group,  to  which  belongs  the  streptothrix  actinomyces, 
or  the  actinomyces  bovis,  and  several  other  important  pathogenic 
agents.  Here  the  organism  consists  of  a  felted  mass  of  non- 
septate  filaments,  in  which  true  dichotomous  branching  occurs. 
Under  certain  circumstances  threads  grow  out,  and  produce 
chains  of  coccus-like  bodies  from  which  new  individuals  can  be 
reproduced.  Such  bodies  are  often  referred  to  as  spores,  but 
they  have  not  the  same  staining  reaction's  nor  resisting  powers 
of  so  high  a  degree  as  ordinary  bacterial  spores.  Sometimes,  too, 
the  protoplasm  of  the  filaments  breaks  up  into  bacillus-like 
elements,  which  may  also  have  the  capacity  of  originating  new 
individuals.  In  the  streptothrix  actinomyces  there  may  appear 
a  club-shaped  swelling  of  the  membrane  at  the  end  of  the  fila- 
ment, which  has  by  some  been  looked  on  as  an  organ  of 
fructification,  but  which  is  most  probably  a  product  of  a 
degenerative  change.  The  streptothrix  group,  though  its 
morphology  and  relationships  are  much  disputed,  may  be  looked 
on  as  a  link  between  the  bacteria  on  the  one  hand,  and  the 
lower  fungi  on  the  other.  Like  the  latter,  the  streptothrix  forms 
show  the  felted  mass  of  non-septate  branching  filaments,  which 
is  usually  called  a  mycelium.  On  the  other  hand,  the  breaking 


FOOD  SUPPLY  17 

up  of  the  protoplasm  of  the  streptothrix  into  coccus-  and  bacillus- 
like  forms,  links  it  to  the  other  bacteria. 

GENERAL  BIOLOGY  OF  THE  BACTERIA. 

There  are  five  prime  factors  in  the  growth  of  bacteria  which 
must  be  considered,  namely,  food  supply,  moisture,  relation  to 
gaseous  environment,  temperature,  and  light. 

Food  Supply. — The  bacteria  are  chiefly  found  living  on  the 
complicated  organic  substances  which  form  the  bodies  of  dead 
plants  and  animals,  or  which  are  excreted  by  the  latter  while 
i  li'-y  are  yet  alive.  Seeing  that,  as  a  general  rule,  many  bacteria 
grow  side  by  side,  the  food  supply  of  any  particular  variety  is, 
relatively  to  it,  altered  by  the  growth  of  the  other  varieties 
present.  It  is  thus  impossible  to  imitate  the  complexity  of  the 
natural  food  environment  of  any  species.  The  artificial  media 
used  in  bacteriological  work  may  therefore  be  poor  substitutes 
for  the  natural  supply.  In  certain  cases,  however,  the  conditions 
under  which  wre  grow  cultures  may  be  better  than  the  natural 
conditions.  For  while  one  of  two  species  of  bacteria  growing 
si<lu  by  side  may  favour  the  growth  of  the  other,  it  may  also 
in  certain  cases  hinder  it,  and  therefore,  when  the  latter  is 
grown  alone  it  may  grow  better.  Most  bacteria  seem  to 
produce  exeretions  which  are  unfavourable  to  their  own 
vitality,  for,  when  a  species  is  sown  on  a  mass  of  artificial 
food  medium,  it  does  not  in  the  great  majority  of  cases  go  on 
growing  till  the  food  supply  is  exhausted,  but  soon  ceases  to 
grow.  Effete  products  diffuse  out  into  the  medium  and  prevent 
growth.  Such  diffusion  may  be  seen  when  the  organism  pro- 
duces pigment,  e.y.  b.  pyocyaneus  growing  on  gelatin.  In 
supplying  artificial  food  for  bacterial  growth,  the  general  principle 
ought  to  be  to  imitate  as  nearly  as  possible  the  natural  surround- 
iuu-s,  though  it  is  found  that  there  exists  a  considerable  adapt- 
ability among  organisms.  With  the  pathogenic  varieties  it  is 
usually  found  expedient  to  use  media  derived  from  the  fluids  of 
tin-  animal  body,  and  in  cases  where  bacteria  growing  on  plants 
are  being  studied,  infusions  of  the  plants  on  which  they  grow 
are  frequently  used.  Some  bacteria  can  exist  on  inorganic  food, 
but  most  require  organic  material  to  be  supplied.  Of  the  latter, 
><>me  require  proteid  to  be  present  for  their  proper  nourishment, 
while  others  can  derive  their  nitrogen  from  a  non-proteid  such 
as  asparagin.  All  bacteria  requre  nitrogen  to  be  present  in 
SMiiu-  form,  and  many  require  to  derive  their  carbon  from 
carl. •ihydrat»i>.  Mineral  salts,  especially  sulphates,  chlorides,  and 


18         GENERAL  MORPHOLOGY  AND  BIOLOGY 

phosphates,  and  also  salts  of  iron  are  necessary.  Occasionally 
special  substances  are  needed  to  support  life.  Thus  some 
species,  in  the  protoplasm  of  which  sulphur  granules  occur, 
require  sulphuretted  hydrogen  to  be  present.  In  nature  the 
latter  is  usually  provided  by  the  growth  of  other  bacteria.  When 
the  food  supply  of  a  bacterium  fails,  it  degenerates  and  dies. 
The  proof  of  death  lies  in  the  fact  that  when  it  is  transferred 
to  fresh  and  good  food  supply  it  does  not  multiply.  If  the 
bacterium  forms  spores,  it  may  then  survive  the  want  of  food 
for  a  very  long  time.  It  may  here  be  stated  that  the  reaction 
of  the  food  medium  is  a  matter  of  great  importance.  Most 
bacteria  prefer  a  slightly  alkaline  medium,  and  some,  e.g.  the 
cholera  spirillum,  will  not  grow  in  the  presence  of  the  smallest 
amount  of  free  acid. 

Moisture. — The  presence  of  water  is  necessary  for  the  con- 
tinued growth  of  all  bacteria.  The  amount  of  drying  which 
bacteria  in  the  vegetative  stage  will  resist  varies  very  much  in 
different  species.  Thus  the  cholera  spirillum  is  killed  by  two  or 
three  hours'  drying,  while  the  staphylococcus  pyogenes  aureus 
will  survive  ten  days'  drying,  and  the  bacillus  diphtheriae  still 
more.  In  the  case  of  spores  the  periods  are  much  longer. 
Anthrax  spores  will  survive  drying  for  several  years,  but  here 
again  moisture  enables  them  to  resist  longer  than  when  they  are 
quite  dry.  When  organisms  have  been  subjected  to»such  hostile 
influences,  even  though  they  survive,  it  by  no  means  follows  that 
they  retain  all  their  vital  properties. 

Relation  to  Gaseous  Environment. — The  relation  of  bacteria 
to  the  oxygen  of  the  air  is  such  an  important  factor  in  the  life 
of  bacteria  that  it  enables  a  biological  division  to  be  made  among 
them.  Some  bacteria  will  only  live  and  grow  when  oxygen  is 
present.  To  these  the  title  of  obligatory  aerobes  is  given.  Other 
bacteria  will  only  grow  when  no  oxygen  is  present.  These  are 
called  obligatory  anaerobes.  In  still  other  bacteria  the  presence 
or  absence  of  oxygen  is  a  matter  of  indifference.  This  group 
might  theoretically  be  divided  into  those  which  are  preferably 
aerobes,  but  can  be  anaerobes,  and  those  which  are  preferably 
anaerobes,  but  can  be  aerobes.  As  a  matter  of  fact  such 
differences  are  manifested  to  a  slight  degree,  but  all  such 
organisms  are  usually  grouped  as  facultative  anaerobes,  i.e.  pre- 
ferably aerobic  but  capable  of  existing  without  oxygen.  Ex- 
amples of  obligatory  aerobes  are  b.  proteus  vulgaris,  b.  subtilis  ;  of 
obligatory  anaerobes,  b.  tetani,  b.  oedematis  maligni,  wThile  the 
great  majority  of  pathogenic  bacteria  are  facultative  anaerobes. 
With  regard  to  anaerobes,  hydrogen  and  nitrogen  are  indifferent 


TEMPERATURE  AND  EFFECT  OF  LIGHT         19 

gases.  Many  anaerobes,  however,  do  not  flourish  well  in  an 
•srttnosphere  of  carbon  dioxide.  Very  few  experiments  have 
been  made  to  investigate  the  action  on  bacteria  of  gas  under 
pressure.  A  great  pressure  of  carbon  dioxide  is  said  to  make 
the  b.  anthracis  lose  its  power  of  sporing,  but  it  seems  to  have 
no  effect  on  its  vitality  or  on  that  of  the  b.  typhosus.  In 
the  case  of  the  bacillus  pyocyaneus,  however,  it  is  said  to 
destroy  life. 

Temperature. — For  every  species  of  bacterium  there  is  a 
temperature  at  which  it  grows  best.  This  is  called  the' 
"  optimum  temperature."  '  There  is  also  in  each  case  a 
maximum  temperature  above  which  growth  does  not  take 
place,  and  a  minimum  temperature  below  which  growth  does 
not  take  place.  As  a  general  rule  the  optimum  temperature  is 
about  the  temperature  of  the  natural  habitat  of  the  organism. 
For  organisms  taking  part  in  the  ordinary  processes  of  putrefac- 
tion the  temperature  of  warm  summer  weather  (20°  to  24°  C.) 
may  l>u  taken  as  the  average  optimum,  while  for  organisms 
normally  inhabiting  animal  tissues  35°  to  39°  C.  is  a  fair 
average.  The  lowest  limit  of  ordinary  growth  is  from  12°  to 
14  C.,  and  the  upper  is  from  42°  to  44°  C.  In  exceptional 
>  growth  may  take  place  as  low  as  5°  C.,  and  as  high  as 
70°  C.  Some  organisms  which  grow  best  at  a  temperature  of  from 
60°  to  70°  C.  have  been  isolated  from  dung,  the  intestinal  tract, 
etc.  These  have  been  called  thermophUic  bacteria.  It  is  to 
be  noted  that  while  growth  does  not  take  place  below  or  above 
;i  certain  limit,  it  by  no  means  follows  that  death  takes  place 
•  Hitside  such  limits.  Organisms  can  resist  cooling  below  their 
minimum  or  heating  beyond  their  maximum  without  being 
killed.  Their  vital  activity  is  merely  paralysed.  Especially  is 
this  true  of  the  effect  of  cold  on  bacteria.  The  results  of 
different  olisrrvers  vary;  but  if  we  take  as  an  example  the 
cholera  vibrio,  Koch  found  that  while  the  minimum  temperature 
of  growth  was  16°  C.,  a  culture  might  be  cooled  to  -32°  C. 
without  being  killed.  With  regard  to  the  upper  limit,  few 
ordinary  organisms  in  a  spore-free  condition  will  survive  a 
temjKirature  of  57°  C.,  if  long  enough  applied.  Many  organisms 
lose  some  of  their  properties  when  grown  at  unnatural  temper- 
atures. Thus  many  pathogenic  organisms  lose  their  virulence 
it  grown  aliovc  their  optimum  temperature,  and  some  chromogenic 
tonns  must  <>!'  \\hich  prefer  rather  low  temperatures,  lose  their 
capacity  of  producing  pigment,  </.//.  spirillum  rabmnL 

Effect  of  Light. — Of  recent  years  much  attention  has  been 
paid  to  this  factor  in  the  life  of  bacteria.     Direct  sunlight  is 


20         GENERAL  MORPHOLOGY  AND  BIOLOGY 

found  to  have  a  very  inimical  effect.  It  has  been  found  that 
an  exposure  of  dry  anthrax  spores  for  one  and  a  half  hours 
to  sunlight  kills  them.  When  they  are  .moist,  a  much  longer 
exposure  is  necessary.  Typhoid  bacilli  are  killed  in  about  one 
and  a  half  hours,  and  similar  results  have  been  obtained  with 
many  other  organisms.  In  such  experiments  the  thickness  of 
the  medium  surrounding  the  growth  is  an  important  point. 
Death  takes  place  more  readily  if  the  medium  is  scanty  or  if 
the  organisms  are  suspended  in  water.  Any  fallacy  which 
might  arise  from  the  effect  of  the  heat  rays  of  the  sun  has  been 
excluded,  though  light  plus  heat  is  more  fatal  than  light  alone. 
In  direct  sunlight  it  is  chiefly  the  green,  violet,  and,  it  may  be, 
the  ultra-violet  rays  which  are  fatal.  Diffuse  daylight  has  also 
a  bad  effect  upon  bacteria,  though  it  takes  a  much  longer  ex- 
posure to  do  serious  harm.  A  powerful  electric  light  is  as 
fatal  as  sunlight.  Here,  as  with  other  factors,  the  results  vary 
very  much  with  the  species  under  observation,  and  a  distinction 
must  be  drawn  between  a  mere  cessation  of  growth  and  the 
condition  of  actual  death.  Some  bacteria,  especially  occurring 
on  the  dead  bodies  of  fresh  fish,  are  phosphorescent. 

Conditions  affecting  the  Movements  of  Bacteria. — In  some 
cases  differences  are  observed  in  the  behaviour  of  motile  bacteria, 
contemporaneous  with  changes  in  their  life  history.  Thus,  in 
the  case  of  bacillus  subtilis,  movement  ceases  when  sporulation 
is  about  to  take  place.  On  the  other  hand,  in  the  bacillus  of 
symptomatic  anthrax,  movement  continues  while  sporulation  is 
progressing.  Under  ordinary  circumstances  motile  bacteria 
appear  not  to  be  constantly  moving,  but  occasionally  to  rest.  In 
every  case  the  movements  become  more  active  if  the  temperature 
be  raised.  Most  interest,  however,  attaches  to  the  fact  that 
bacilli  may  be  attracted  to  certain  substances  and  repelled  by 
others.  Schenk,  for  instance,  observed  that  motile  bacteria 
were  attracted  to  a  warm  point  in  a  way  which  did  not  occur 
when  the  bacteria  were  dead  and  therefore  only  subject  to 
physical  conditions.  Most  important  observations  have  been 
made  on  the  attraction  and  repulsion  exercised  on  bacteria  by 
chemical  agents,  which  have  been  denominated  respectively 
positive  and  '  negative  chemiotaxis.  Pfeffer  investigated  this 
subject  in  many  lowly  organisms,  including  bacterium  termo 
and  spirillum  undula.  The  method  was  to  fill  with  the  agent 
a  fine  capillary  tube,  closed  at  one  end,  to  introduce  this  into 
a  drop  of  fluid  containing  the  bacteria  under  a  cover-glass,  and 
to  watch  the  effect  through  the  microscope.  The  general  result 
was  to  indicate  that  motile  bacteria  may  be  either  attracted  or 


THE  PARTS  PLAYED  BY  BACTERIA  IN  NATURE     21 

repelled  by  the  fluid  in  the  tube.  The  effect  of  a  given  fluid 
differs  in  different  organisms,  and  a  fluid  chemiotactic  for  one 
organism  may  not  act  on  another.  Degree  of  concentration  is 
important,  but  the  nature  of  the  fluid  is  more  so.  Of  inorganic 
bodies  salts  of  potassium  are  the  most  powerfully  attracting 
bodies,  and  in  comparing  organic  bodies  the  important  factor 
is  the  molecular  constitution.  These  observations  have  been 
confirmed  by  Ali-Cohen,  who  found  that  while  the  vibrio  of 
cholera  and  the  typhoid  bacillus  were  scarcely  attracted  by 
chloride  of  potassium,  they  were  powerfully  influenced  by 
potato  juice.  Further,  the  filtered  products  of  the  growth  of 
many  bacteria  have  been  found  to  have  powerful  chemiotactic 
properties.  It  is  evident  that  all  these  observations  have  a 
most  important  bearing  on  the  action  of  bacteria,  though  we 
do  not  yet  know  their  true  significance.  Corresponding  chemio- 
tactic phenomena  are  shown  also  by  certain  animal  cells,  e.g. 
leucocytes,  to  which  reference  is  made  below. 

The  Parts  played  by  Bacteria  in  Nature. — As  has  been  said, 
tlic  chief  effect  of  bacterial  action  in  nature  is  to  break  up  into 
more  simple  combinations  the.  complex  molecules  of  the  organic 
substances  which  form  the  bodies  of  plants  and  animals,  or 
which  are. excreted  by  them.  In  some  cases  we  know  some  of 
the  stages  of  disintegration,  but  in  most  cases  we  know  only 
general  principles  and  sometimes  only  results.  In  the  case  of 
milk,  for  instance,  we  know  that  lastjc  acid  is  produced  from 
t,frfi  lap.f-Qfip,  by  the  action  of  the  bacillus  acidi  lactici  and  of 
other  bacteria,  and  that  from  urea  ammonium  carbonate  is 
produced  by  the  micrococcus  ureae.  That  the  very  complicated 
process  of  putrefaction  is  due  to  bacteria  is  absolutely  proved, 
for  any  organic  substance .  can  be  preserved  indefinitely  from 
ordinary  putrefaction  by  the  adoption  of  some  method  of 
killing  all  bacteria  present  in  it,  as  will  be  afterwards  described. 
This  statement,  however,  does  not  exclude  the  fact  that 
molecular  changes  take  place  spontaneously  in  the  passing  of 
tlit-  organic  body  from  life  to  death.  Many  processes  not 
usually  referred  to  as  putrefactive  are  also  bacterial  in  their 
origin.  The  souring  of  milk,  already  referred  to,  the  becoming 
rancid  of  butter,  the  ripening  of  cream  and  of  cheese,  are  all 
due  to  bacteria. 

A  certain  comparatively  small  number  of  bacteria  have  been 
I -loved  to  be  the  causal  agents  in  some  disease  processes 
occurring  in  man,  animals,  and  plants.  This  means  that  the 
fluids  and  tissues  of  living  bodies  are,  under  certain  circum- 
>tauces,  a  suitable  pabulum  for  the  bacteria  involved.  The 


22         GENERAL  MORPHOLOGY  AND  BIOLOGY 

effects  of  the  action  of  these  bacteria  are  analogous  to  those 
taking  place  in  the  action  of  the  same  or  other  bacteria  on  dead 
animal  or  vegetable  matter.  The  complex  organic  molecules 
are  broken  up  into  simpler  products.  We  shall  study  these 
processes  more  in  detail  later.  Meantime  we  may  note  that 
the  disease-producing  effects  of  bacteria  form  the  basis  of 
another  biological  division  of  the  group.  Some  bacteria  are 
harmless  to  animals  and  plants,  and  apparently  under  no 
circumstances  give  rise  to  disease  in  either.  These  are  known 
as  saprophytes.  They  are  normally  engaged  in  breaking  up 
dead  animal  and  vegetable  matter.  Others  normally  live  on 
or  in  the  bodies  of  plants  and  animals  and  produce  disease. 
These  are  known  as  parasitic  bacteria.  Sometimes  an  attempt 
is  made  to  draw  a  hard-and-fast  line  between  the  saprophytes 
and  the  parasites,  and  obligatory  saprophytes  or  parasites  are 
spoken  of.  This  is  an  erroneous  distinction.  Some  bacteria 
which  are  normally  saprophytes  can  produce  pathogenic  effects 
(e.g.  bacillus  oedematis  maligni),  and  it  is  consistent  with  our 
knowledge  that  the  best-known  parasites  may  have  been  derived 
from  saprophytes.  On  the  other  hand,  the  fact  that  most 
bacteria  associated  with  disease  processes,  and  proved  to  be 
the  cause  of  the  latter,  can  be  growTi  in  artificial  media,  show's 
that  for  a  time  at  least  such  parasites  can  be  saprophytic.  As 
to  how  far  such  a  saprophytic  existence  of  disease-producing 
bacteria  occurs  in  nature,  we  are  in  many  instances  still 
ignorant. 

The  Methods  of  Bacterial  Action. — The  processes  which 
bodies  undergo  in  being  split  up  by  bacteria  depend,  first,  on 
the  chemical  nature  of  the  bodies  involved,  and,  secondly,  on 
the  varieties  of  the  bacteria  which  are  acting.  The  destruction 
of  albuminous  bodies  which  is  mostly  involved  in  the  wide  and 
varied  process  of  putrefaction  can  be  undertaken  by  whole 
groups  of  different  varieties  of  bacteria.  The  action  of  the 
latter  on  such  substances  is  analogous  to  what  takes  place  when 
albumins  are  subjected  to  ordinary  gastric  and  intestinal 
digestion.  In  these  circumstances,  therefore,  the  production 
of  albumoses,  peptones,  etc.,  similar  to  those  of  ordinary 
digestion,  can  be  recognised  in  putrefying  solutions,  though 
the  process  of  destruction  always  goes  further,  and  still  simpler 
substances,  e.g.  indol,  and,  it  may  be,  crystalline  bodies  of  an 
alkaloidal  nature,  are  the  ultimate  results.  The  process  is 
an  exceedingly  complicated  one  when  it  takes  place  in  nature, 
and  different  bacteria  are  probably  concerned  in  the  different 
stages.  Many  other  bacteria,  e.g.  some  pathogenic  forms, 


THE  METHODS  OF  BACTERIAL  ACTION         23 

though  not  concerned  in  ordinary  putrefactive  processes,  have 
a  similar  digestive  rapacity.  When  carbohydrates  are  being 
split  up,  then  various  alcohols,  ethers,  and  acids  are  produced. 
l)uring  bacterial  growth  there  is  not  infrequently  the  abundant 
production  of  such  gases  as  sulphuretted  hydrogen,  carbon 
dioxide,  methane,  etc..  For  an  exact  knowledge  of  the  de- 
structive capacities  of  any  particular  bacterium  there  must  be 
an  accurate  chemical  examination  of  its  effects  when  it  has 
been  grown  in  artificial  media  the  nature  of  which  is  known. 
The  precise  substances  it  is  capable  of  forming  can  thus  be 
found  out.  Many  substances,  however,  are  produced  by 
bacteria,  of  the  exact  nature  of  which  we  are  still  ignorant, 
for  example,  the  toxic  bodies  which  play  such  an  important 
part  in  the  action  of  many  pathogenic  species. 

Many  of  the  actions  of  bacteria  depend  on  the  production  by 
them  si  ferments  of  a  very  varied  nature  and  complicated  action. 
Thus  the  digestive  action  on  albumins  probably  depends  on  the 
production  of  a  peptic  ferment  analogous  to  that  produced  in  the 
animal  stomach.  Ferments  which  invert  sugar,  which  .split 
up  sugars  into  alcohols  or  acids,  which  coagulate  casein,  which 
split  up  mva  into  ammonium  carbonate,  also  occur. 

Such  ferments  may  be  diffused  into  the  surrounding  fluid,  or 
be  retained  in  the  cells  where  they  are  formed.  Sometimes  the 
breaking  down  of  the  organic  matter  appears  to  take  place 
within,  or  in  the  immediate  proximity  of,  the  bacteria,  some- 
times wherever  the  soluble  ferments  reach  the  organic  substances. 
And  in  certain  cases  the  ferments  diffusing  out  into  the  surround- 
ing medium  probably  break  down  the  constituents  of  the  latter 
t->  some  extent,  and  prepare  them  for  a  further,  probably 
intracellular,  disintegration.  Thus,  in  certain  putrefactions  of 
fibrin,  if  the  process  be  allowed  to  go  on  naturally,  the  fibrin 
dissolves  and  ultimately  great  gaseous  evolution  of  carbon 
di«>xidf  and  ammonia  takes  place,  but  if  the  bacteria,  shortly 
after  the  process  has  begun,  are  killed  or  paralysed  by  chloro- 
form, then  only  a  peptonisation  of  the  fibrin  occurs,  without 
the  further  splitting  up  and  gaseous  production.  That  a 
purely  intracellular  digestion  may  take  place  is  illustrated  by 
what  has  been  shown  to  occur  in  the  case  of  the  micrccoccus 
urea-,  which  from  urea  forms  ammonium  carbonate  by  adding 
water  to  the  urea  molecule.  Here,  if  after  the  action  has 
commenced  the  bacteria  are  filtered  off,  no  further  production 
of  ammonium  carbonate  takes  place,  which  shows  that  no 
ferment  has  been  dissolved  out  into  the  urine.  If  now  the 
bodies  of  the  bacteria  be  extracted  with  absolute  alcohol  or  ether, 


24         GENERAL  MORPHOLOGY  AND  BIOLOGY 

which  of  course  destroy  their  vitality,  a  substance  is  obtained 
of  the  nature  of  a  ferment,  which,  when  added  to  sterile  urine, 
rapidly  causes  the  production  of  ammonium  carbonate.  This 
ferment  has  evidently  been  contained  within  the  bacterial  cells. 
In  the  investigation  of  the  phenomena  of  the  ferment  action  of 
bacteria,  it  has  been  noted  in  certain  cases  that  the  ferments 
formed  depend  on  the  food  supply  offered  to  the  bacterium. 
Thus  in  one  case  a  bacterium  growing  in  starch  forms  diastase, 
which  it  does  not  do  when  grown  on  sugar. 

In  considering  the  effects  of  bacteria  in  nature,  it  must  be  recognised 
that  some  species  are  capable  of  building  up  complex  substances  out  of 
simple  chemical  compounds.  Examples  of  these  are  found  in  the  bacteria 
which  in  the  soil  make  nitrogen  more  available  for  plant  nutrition  by 
converting  ammonia  into  nitrites  and  nitrates.  Winogradski,  by  using 
media  containing  non-nitrogenous  salts  of  magnesium,  potassium,  and 
ammonium,  and  free  of  organic  matter,  has  demonstrated  the  existence 
of  forms  which  convert,  by  oxidation,  ammonia  into  nitrites,  and  of  other 
forms  which  convert  these  nitrites  into  nitrates.  Both  can  derive  their 
necessary  carbon  from  alkaline  carbonates.  Other  bacteria,  or  organisms 
allied  to  the  bacteria,  exist  which  can  actually  take  up  and  combine  into 
new  compounds  the  free  nitrogen  of  the  air.  These  are  found  in  the 
tubercles  which  develop  on  the  rootlets  of  the  leguminosre.  Without 
such  organisms  the  tubercles  do  not  develop,  and  without  the  development 
of  the  tubercles  the  plants  are  poor  and  stunted.  Bacteria  thus  play  an 
important  part  in  the  enrichment  and  fertilisation  of  the  soil. 

The  Occurrence  of  Variability  among  Bacteria.  — The  question  of  the 
division  of  the  group  of  bacteria  into  definite  species  has  given  rise  to 
much  discussion  among  vegetable  and  animal  morphologists,  and  at  one 
time  very  divergent  views  were  held.  Some  even  thought  that  the 
same  species  might  at  one  time  give  rise  to  one  disease, — at  another  time 
to  another.  There  is,  however,  now  practical  unanimity  that  bacteria 
show  as  distinct  species  as  the  other  lower  plants  and  animals,  though, 
of  course,  the  difficulty  of  defining  the  concept  of  a  species  is  as  great  in 
them  as  it  is  in  the  latter.  Still,  we  can  say  that  among  the  bacteria  AVC 
see  exhibited  (to  use  the  words  of  De  Bary)  "the  same  periodically 
repeated  course  of  development  within  certain  empirically  determined 
limits  of  variation  "  which  justifies,  among  higher  forms  of  life,  a  species 
to  be  recognised.  What  at  first  raised  doubts  as  to  the  occurrence  of 
species  among  the  bacteria  was  the  observation  in  certain  cases  of  what  is 
known  as  pleomorpliism.  By  this  is  meant  that  one  species  may  assume 
at  different  times  different  forms,  e.g.  appear  as  a  coccus,  a  bacillus,  or 
a  leptothrix.  Undoubtedly,  many  of  the  cases  where  this  was  alleged 
to  have  been  observed  occurred  before  the  elaboration  of  the  modern 
technique  for  the  obtaining  of  pure  cultures,  but  at  the  present  day  there 
are  cases  where  evidence  appears  to  exist  of  the  occurrence  of  pleomorphism. 
This  is  especially  the  case  with  certain  bacilli,  and  it  may  lead  to  such 
forms  being  classed  among  the  higher  bacteria.  Pleomorphism  is, 
however,  a  rare  condition,  and  with  regard  to  the  bacteria  as  a  whole  we 
may  say  that  each  variety  tends  to  conform  to  a  definite  type  of  structure 
and  function  which  is  peculiar  to  it  and  to  it  alone.  On  the  other  hand, 
slight  variations  from  such  type  can  occur  in  each.  The  size  may  vary 


VARIABILITY  AMONG  BACTERIA  25 

a  little  with  the  medium  in  which  the  organism  is  growing,  and  under 
certain  similar  conditions  the  adhesion  of  bacteria  to  each  other  may  also 
vary.  Thus  cocci,  which  are  ordinarily  seen  in  short  chains,  may  grow 
in  long  chains.  The  capacity  to  form  spores  may  be  altered,  and  such 
properties  as  the  elaboration  of  certain  ferments  or  of  certain  pigments 
may  be  impaired.  Also  the  characters  of  the  growths  on  various  media 
may  undergo  variations.  As  has  been  remarked,  variation  as  observed 
consists  largely  in  a  tendency  in  a  bacterium  to  lose  properties  ordinarily 
possessed,  and  all  attempts  to  transform  one  bacterium  into  an  apparently 
closely  allied  variety  (such  as  the  b.  coli  into  the  b.  typhosus)  have 
failed.  This  of  course  does  not  preclude  the  possibility  of  one  species 
having  been  originally  derived  from  another,  or  of  both  having  descended 
from  a  common  ancestor,  but  we  can  say  that  only  variations  of  an 
unimportant  order  have  been  observed  to  take  place,  and  here  it  must  be 
remembered  that  in  many  cases  we  can  have  forty-eight  or  more 
generations  under  observation  within  twenty-four  hours. 


CHAPTER   IT. 

METHODS  OF  CULTIVATION  OF  BACTERIA. 

Introductory. — In  order  to  study  the  characters  of  any  species 
of  bacterium,  it  is  necessary  to  have  it  growing  apart  from  every 
other  species.  In  the  great  majority  of  cases  where  bacteria 
occur  in  nature,  this  condition  is  not  fulfilled.  Only  in  the 
blood  and  tissues  in  some  diseases  do  particular  species  occur 
singly  and  alone.  We  usually  have,  therefore,  to  remove  a 
bacterium  from  its  natural  surroundings  and  grow  it  on  an 
artificial  food  medium.  When  we  have  succeeded  in  separating 
it,  and  have  got  it  to  grow  on  a  medium  which  suits  it,  wre  are 
said  to  have  obtained  a  pure  culture.  The  recognition  of 
different  species  of  bacteria  depends,  in  fact,  far  more  on  the 
characters  presented  by  pure  cultures  and  their  behaviour  in 
different  food  media,  than  on  microscopic  examination.  The 
latter  in  most  cases  only  enables  us  to  refer  a  given  bacterium 
to  its  class.  Again,  in  inquiring  as  to  the  possible  possession  of 
pathogenic  properties  by  a  bacterium,  the  obtaining  of  pure 
cultures  is  absolutely  essential. 

To  obtain  pure  cultures,  then,  is  the  first  requisite  of  bacterio- 
logical research.  Now,  as  bacteria  are  practically  omnipresent, 
we  must  first  of  all  have  means  of  destroying  all  extraneous 
organisms  which  may  be  present  in  the  food  media  to  be  used 
in  the  vessels  in  which  the  food  media  are  contained,  and  on  all 
instruments  which  are  to  come  in  contact  with  our  cultures. 
The  technique  of  this  destructive  process  is  called  sterilisation. 
We  must  therefore  study  the  methods  of  sterilisation.  The 
growth  of  bacteria  in  other  than  their  natural  surroundings 
involves  further  the  preparation  of  sterile  artificial  food  media, 
and  when  we  have  such  media  prepared  we  have  still  to  look 
at  the  technique  of  the  separation  of  micro-organisms  from 
mixtures  of  these,  and  the  maintaining  of  pure  cultures  when  the 
latter  have  been  obtained.  We  shall  here  find  that  different 
methods  are  necessary  according  as  we  are  dealing  with  aerobes 

26 


STERILISATION  BY  DRY  HEAT  27 

or  anaerobes.      Each    of  these    methods   will    l>u    considered  in 

turn. 

THE  METHODS  OF  STERILISATION. 

To  exclude  extraneous  organisms,  all  food  materials,  glass 
vessels  containing  them,  wires  used  in  transferring  bacteria  from 
one  culture  medium  to  another,  instruments  used  in  making 
autopsies,  etc.,  must  be  sterilised.  These  objects  being  so 
different,  various  methods  are  necessary,  but  underlying  these 
methods  is  the  general  principle  that  all  bacteria  are  destroyed 
by  heat.  The  temperature  necessary  varies  with  different 
bacteria,  and  the  vehicle  of  heat  is  also  of  great  importance. 
The  two  vehicles  employed  are  hot  air  and  hot  water  or  steam. 
The  former  is  usually  referred  to  as  "  dry  heat,"  the  latter  as 
"moist  heat."  As  showing  the  different  effects  of  the  two 
vehicles,  Koch  found,  for  instance,  that  the  spores  of  bacillus 
anthraois,  which  were  killed  by  moist  heat  at  100°  C.,  in  one 
hour,  required  three  hours'  dry  heat  at  140°  C.  to  effect  death. 
Both  forms  of  heat  may  be  applied  at  different  temperatures — 
in  the  case  of  moist  heat  above  100°  C.,  a  pressure  higher  than 
that  of  the  atmosphere  must  of  course  be  present. 

A.  Sterilisation  by  Dry  Heat. 

A.  (1)  Red  Heat  or  Dull  Red  Heat. — Red  heat  is  used  for 
the  sterilisation  of  the  platinum  needles  which,  it  will  be  found, 
are  so  constantly  in  use.  A  dull  heat  is  used  for  cauteries,  the 
I  mints  of  forceps,  and  maybe  used  for  the  incidental  sterilisation 
of  small  glass  objects  (cover-slips,  slides,  occasionally  when 
necessary  even  test-tubes),  care  of  course  being  taken  not  to 
melt  the  glass.  The  heat  is  obtained  by  an  ordinary  Bunsen 
burner. 

A.  ('!)  Sterilisation  by  Dry  Heat  in  a  Hot- Air  Chamber.— 
The  chamber  (Fig.  2)  consists  of  an  outer  and  inner  case  of 
sheet  iron.  In  the  bottom  of  the  outer  there  is  a  large  hole. 
A  Bunsen  is  lit  beneath  this,  and  thus  plays  on  the  bottom  of 
the  inner  case,  round  all  the  sides  of  which  the  hot  air  rises 
and  escapes  through  holes  in  the  top  of  the  outer  case.  A 
thermometer  passes  down  into  the  interior  of  the  chamber,  half- 
way up  which  its  bulb  should  be  situated.  It  is  found,  as  a 
matter  of  experience,  that  an  exposure  in  such  a  chamber  for 
one  hour  to  a  temperature  of  160°  C.,  is  sufficient  to  kill  all  the 
organisms  which  usually  pollute  articles  in  a  bacteriological 
laboratory,  though  circumstances  might  arise  where  this  would 


28      METHODS  OF  CULTIVATION  OF  BACTERIA 


be  insufficient. 


This  means  of  sterilisation  is  used  for  the  glass 
flasks,  test-tubes,  plates,  Petri's 
dishes,  the  use  of  which  will 
be  described.  Such  pieces  of 
apparatus  are  thus  obtained 
sterile  and  dry.  It  is  advisable 
to  put  glass  vessels  into  the 
chamber  before  heating  it,  and 
to  allow  them  to  stand  in  it 
after  sterilisation  till  the  tem- 
perature falls.  Sudden  heating 
or  cooling  is  apt  to  cause 
glass  to  crack.  The  method  is 
manifestly  unsuitable  for  food 
media. 

B.  Sterilisation  by  Moist 
Heat. 


FIG.  2.—  Hot-air  steriliser.  B.     (1)    By    Boiling.  —  The 

boiling  of   a    liquid    for    five 

minutes  is  sufficient  to  kill  ordinary  germs  if  no  spores  be 
present,  and  this  method  is  useful  for  sterilising  distilled  or  tap 
water  which  may  be  required  in  various  manipulations.  It  is 
best  to  sterilise  knives  and  instruments 
used  in  autopsies  by  boiling  in  water  to 
which  a  little  sodium  carbonate  has  been 
added  to  prevent  rusting.  Twenty  minutes' 
boiling  will  here  be  sufficient.  The  boiling 
of  any  fluid  at  100°  C.  for  one  and  a  half 
hours  will  ensure  sterilisation  under  almost 
any  circumstances. 

B.  (2)  By  Steam  at  100°  C.—  This  is  by 
far  the  most  useful  means  of  sterilisation. 
It  may  be  accomplished  in  an  ordinary 
potato  steamer  placed  on  a  kitchen  pot. 
The  apparatus  ordinarily  used  is  "Koch's 
steam  steriliser  "  (Fig.  3).  This  consists  of 
a  tall  metal  cylinder  on  legs,  provided  with 
a  lid,  and  covered  externally  by  some  bad 
conductor  of  heat,  such  as  felt  or  asbestos. 
A  perforated  tin  diaphragm  is  fitted  in 

the  interior  at  a  little   distance  above  the  ™T          ^    ,  , 

T,  ,    ,,  .       ,  FIG.  3.  —  Koch  s  steam 

bottom,  and  there  is  a  tap  at  the  bottom  steriliser. 


STERILISATION  BY  STEAM  29 

by  which  water  may  be  supplied  or  withdrawn.  If  water 
to  the  depth  of  3  inches  be  placed  in  the  interior  and  heat 
applied,  it  will  quickly  boil,  and  the  steam  streaming  up  will 
surround  any  flask  or  other  object  standing  on  the  diaphragm. 
Here  no  evaporation  takes  place  from  any  medium,  as  it  is  sur- 
rounded during  sterilisation  by  an  atmosphere  saturated  with 
\\atrr  vapour.  It  is  convenient  to  have  the  cylinder  tall  enough 
to  hold  a  litre  flask  with  a  funnel  7  inches  in  diameter  standing 
in  its  neck.  The  funnel  may  be  supported  by  passing  its  tube 
through  a  second  perforated  diaphragm  placed  in  the  upper  part 
of  the  steam  chamber.  With  such  a  "  Koch  "  in  the  laboratory 
a  hot- water  filter  is  not  needed.  As  has  been  said,  one  and  a 
half  hour's  steaming  will  sterilise  any  medium,  but  in  the  case 
of  media  containing  gelatin  such  an  exposure  is  not  practicable, 
as,  with  long  boiling,  gelatin  tends  to  lose  its  physical  property 
of  solidification.  The  method  adopted  in  this  case  is  to  steam 
for  twenty  minutes  on  each  of  three  succeeding  days. 

This  is  ;i  modification  of  what  is  known  as  "Tyndall's  intermittent 
>t<  rilisation."  The  fundamental  principle  of  this  method  is  that  all 
ba'-tt'ria  in  a  non-spored  form  are  killed  by  the  temperature  of  boiling 
\\ater.  while  if  in  a  spored  form  they  may  not  be  thus  killed.  Thus  by 
the  sterilisation  mi  the  first  day  all  the  non-spored  forms  are  destroyed — 
the  spores  remaining  alive.  During  the  twenty-four  hours  which  intervene 
before  the  next  heating,  these  spores,  being  in  a  favourable  medium,  are 
likely  to  assume  the  non-spored  form.  The  next  heating  kills  these.  In 
case  any  may  still  not  have  changed  their  spored  form,  the  process  is 
repeated  on  a  third  day.  Experience  shows  that  usually  the  medium 
can  now  be  kept  indefinitely  in  a  sterile  condition. 

Steam  at  100°  C.  is  therefore  available  for  the  sterilisation  of 
all  ordinary  media.  In  using  the  Koch's  steriliser,  especially 
wlu-n  a  large  bulk  is  to  be  sterilised,  it  is  best  to  put  the  medium 
in  while  the  apparatus  is  cold,  in  order  to  make  certain  that  the 
whole  of  the  food  mass  reaches  the  temperature  of  100°  C.  The 
I'fiiod  of  exposure  is  reckoned  from  the  time  boiling  commences 
in  the  water  in  the  steriliser.  At  any  rate  allowance  must 
always  In-  made  for  the  time  required  to  raise  the  temperature 
of  the  medium  to  that  of  the  steam  surrounding  it. 

B.  (:\)  Sterilisation  by  Steam  at  High  Pressure. — This  is 
the  most  rapid  and  effective  means  of  sterilisation.  It  is  effected 
in  an  autoclave  (Fig.  4).  This  is  a  gun-metal  cylinder  supported 
in  a  cylindrical  sheet-iron  case ;  its  top  is  fastened  down  with 
screws  and  nuts,  and  is  furnished  with  a  safety  valve,  pressure- 
^aiige,  and  a  hole  for  thermometer.  As  in  the  Koch's  steriliser, 
the  contents  are  supported  on  a  perforated  diaphragm.  The 


30     METHODS  OF  CULTIVATION  OF  BACTERIA 


0  0  o 


oooo 


source  of  heat  is  a  large  Bunsen  beneath.  The  temperature 
employed  is  usually  115°  C.  or  120°  C.  To  boil  at  115°  C., 
water  requires  a  pressure  of  about  23  Ibs.  to  the  square  inch 
(i.e.  8  Ibs.  plus  the  15  Ibs.  of  ordinary  atmo- 
spheric pressure).  To  boil  at  120°  C.,  a 
pressure  of  about  30  Ibs.  (i.e.  15  Ibs.  plus 
the  usual  pressure)  is  necessary.  In  such 
an  apparatus  the  desired  temperature  is 
maintained  by  adjusting  the  safety-valve  so 
as  to  blow  off  at  the  corresponding  pressure. 
One  exposure  of  media  to  such  temperatures 
for  a  quarter  of  an  hour  is  amply  sufficient 
to  kill  all  organisms  or  spores.  Here,  again, 
care  must  be  taken  when  gelatin  is  to  be 
sterilised.  It  must  not  be  exposed  to  a 
temperature  above  105°  C.,  and  is  best 
sterilised  by  the  intermittent  method. 
Certain  precautions  are  necessary  in  using 
the  autoclave.  In  all  cases  it  is  necessary 
to  allow  the  apparatus  to  cool  well  below 
100°  C.  before  opening  it  or  allowing  steam 
to  blow  off,  otherwise  there  will  be  a  sudden 
development  of  steam  when  the  pressure  is 
removed,  and  fluid  media  will  be  blown  out 
of  the  flasks.  Sometimes  the  instrument  is 
not  fitted  with  a  thermometer.  In  this  case  care  must  be 
taken  to  expel  all  the  air  initially  present,  otherwise,  a  mixture 
of  air  and  steam  being  present,  the  pressure  read  off  the  gauge 
cannot  be  accepted  as  an  indication  of  the  temperature.  Further, 
care  must  be  taken  to  ensure  the  presence  of  a  residuum  of 
water  when  steam  is  fully  up,  otherwise  the  steam  is  super- 
heated, and  the  pressure  on  the  gauge  again  does  not  indicate 
the  temperature  correctly. 

B.  (4)  Sterilisation  at  Low  Temperatures. — Most  organisms 
in  a  non-spored  form  are  killed  by  a  prolonged  exposure  to  a 
temperature  of  57°  C.  This  fact  has  been  taken  advantage  of 
for  the  sterilisation  of  blood  serum,  which  will  coagulate  if 
exposed  to  a  temperature  above  that  point.  Such  a  medium  is 
sterilised  on  Tyndall's  principle  by  exposing  it  for  an  hour  at 
57°  C.  for  eight  consecutive  days,  it  being  allowed  to  cool  in  the 
interval  to  the  room  temperature.  The  apparatus  shown  in 
Fig.  5  is  a  small  hot-water  jacket  heated  by  a  Bunsen  placed 
beneath  it,  the  temperature  being  controlled  by  a  gas  regulator. 
To  ensure  that  the  temperature  all  around  shall  be  the  same, 


FIG.  4. — Autoclave. 

a.  Safety-valve. 
6.  Blow-off  pipe. 
c.  Gauge. 


PREPARATION  OF  ORDINARY  CULTURE  MEDIA     31 


the  lid  also  is  hollow  and  filled  with  water,  and  there  is  a 
special  gas  burner  at  the  side  to  heat  it.  This  is  the  form 
originally  used,  but  serum  sterilisers  are  now  constructed  in 
which  the  test-tubes  are  placed  iu 


the 


position,    and   in    which 


inspissatiori  (vide  p.  40)  can  after- 
wards be  performed  at  a  higher 
temperature. 

THE  PREPARATION  OF  ORDINARY 
CULTURE  MEDIA. 

The  general  principle  to  be  observed 
in  the  artificial  culture  of  bacteria  is 
that  the  medium  used  should  approxi- 
mate as  closely  as  possible  to  that  on 
which  the  bacterium  growrs  naturally. 
In  the  case  of  pathogenic  bacteria  the 
medium  therefore  should  resemble  the 
juices  of  the  body.  The  serum  of 
the  blood  satisfies  this  condition,  and 
is  often  used,  but  its  application  is 

limited     by    the     difficulties     in     its     F]G.  5._steriliser  for  blood 
preparation   and  preservation.     Other  serum. 

media    have    been    found    which    can 

support  the  life  of  all  the  pathogenic  bacteria  isolated.  These 
consist  of  proteids  or  carbohydrates  in  a  fluid,  semi-solid, 
or  solid  form,  in  a  transparent  or  opaque  condition.  The 
advantage  of  having  a  variety  of  media  lies  in  the  fact  that 
growth  characters  on  particular  media,  non-growth  on  some 
and  growth  on  others,  etc.,  constitute  specific  differences 
which  are  valuable  in  the  identification  of  bacteria.  The 
most  commonly  used  media  have  as  their  basis  a  watery 
extract  of  meat.  Most  bacteria  in  growing  in  such  an 
extract  cause  only  a  grey  turbidity.  A  great  advance  resulted 
when  Koch,  by  adding  to  it  gelatin,  provided  a  transparent 
solid  medium  in  which  growth  characteristics  of  particular 
bacteria  become  evident.  Many  organisms,  however,  grow  best 
at  a  temperature  at  which  this  nutrient  gelatin  is  fluid,  and 
therefore  another  gelatinous  substance  called  agar,  which  does 
not  melt  below  98°  C.,  was  substituted.  Bouillon  made  from 
Mirat  extract,  gelatin,  and  agar  media,  and  the  modifications 
of  these,  constitute  the  chief  materials  in  which  bacteria  are 
grown. 


32      METHODS  OF  CULTIVATION  OF  BACTERIA 

Preparation  of  Meat  Extract. 

The  flesh  of  the  ox,  calf,  or  horse  is  usually  employed. 
Horse-flesh  has  the  advantage  of  being  cheaper  and  containing 
less  fat  than  the  others ;  though  generally  quite  suitable,  it  has 
the  disadvantage  for  certain  purposes  of  containing  a  larger 
proportion  of  fermentable  sugar.  The  flesh  must  be  freed  from 
fat,  and  finely  minced.  To  a  pound  of  mince  add  1000  c.c. 
distilled  water,  and  mix  thoroughly  in  a  shallow  dish.  Set 
aside  in  a  cool  place  for  twenty-four  hours.  Skim  off  any  fat 
present,  removing  the  last  traces  by  stroking  the  surface  of  the 
fluid  with  pieces  of  filter  paper.  Place  a  clean  linen  cloth  over 
the  mouth  of  a  large  filter  funnel,  and  strain  the  fluid  through 
it  into  a  flask.  Pour  the  minced  meat 
into  the  cloth,  and,  gathering  up  the 
edges  of  the  latter  in  the  left  hand, 
squeeze  out  the  juice  still  held  back  in 
the  contained  meat.  Finish  this  expres- 
sion by  putting  the  cloth  and  its  contents 
into  a  meat  press  (Fig.  6),  similar  to 
that  used  by  pharmacists  in  preparing 
extracts  ;  thus  squeeze  out  the  last  drops. 
The  resulting  sanguineous  fluid  contains 
the  soluble  albumins  of  the  meat,  the 
soluble  salts,  extractives,  and  colouring 
matter,  chiefly  haemoglobin.  It  is  now 
FIG.  6.— Meat  press.  boiled  thoroughly  for  two  hours,  by 
which  process  the  albumins  coagulable 

by  heat  are  coagulated.  Strain  now  through  a  clean  cloth, 
boil  for  another  half-hour,  and  filter  through  white  Swedish 
filter  paper  (best,  C.  Schleicher .  u.  Schull,  No.  595).  Make 
up  to  1000  c.c.  with  distilled  water.  The  resulting  fluid 
ought  to  be  quite  transparent,  of  a  yellowish  colour  without 
any  red  tint.  If  there  is  any  redness,  the  fluid  must  be 
reboiled  and  filtered  till  this  colour  disappears,  otherwise  in 
the  later  stages  it  will  become  opalescent.  A  large  quantity 
of  the  extract  may  be  made  at  a  time,  and  what  is  not 
immediately  required  is  put  into  a  large  flask,  the  neck  plugged 
with  cotton  wool,  and  the  whole  sterilised  by  methods  B  (2)  or 
(3).  This  extract  contains  very  little  albuminous  matter,  and 
consists  chiefly  of  the  soluble  salts  of  the  muscle,  certain 
extractives,  and  altered  colouring  matters,  along  with  any  slight 
traces  of  soluble  proteid  not  coagulated  by  heat.  It  is  of  acid 
reaction.  We  have  now  to  see  how,  by  the  addition  of  proteid 


BOUILLON  MEDIA  33 

and  other  matter,  it  may  be  transformed  into  proper  culture 
media. 

1.  Bouillon  Media.— These  consist  of  meat  extract  with  the 
addition  of  certain  substances  to  render  them  suitable  for  the 
growth  of  bacteria. 

(1)  (a).  Peptone  Broth  or  Bouillon. — This  has  the  com- 
position : — 

Meat  extract1    ....        1000  c.c. 
Sodium  chloride         ...  5  grms. 

Peptone  albumin        .          .          .  10     „ 

Boil  till  the  ingredients  are  quite  dissolved,  and  neutralise 
with  a  saturated  solution  of  sodium  hydrate.  Add  the  latter 
drop  by  drop,  shaking  thoroughly  between  each  drop  and  testing 
the  reaction  by  means  of  litmus  paper.  Go  on  till  the  reaction 
is  slightly  but  distinctly  alkaline.  Neutralisation  must  be 
practised  with  great  care,  as  under  certain  circumstances, 
depending  on  the  relative  proportions  of  the  different  phosphates 
of  sodium  and  potassium,  what  is  known  as  the  amphoteric 
reaction  is  obtained,  i.e.  red  litmus  is  turned  blue,  and  blue  red, 
by  the  same  solution.  The  sodium  hydrate  must  be  added  till 
red  litmus  is  turned  slightly  but  distinctly  blue,  and  blue  litmus 
is  not  at  all  tinted  red.  After  alkalinisation,  allow  the  fluid  to 
become  cold,  filter  through  Swedish  filter  paper  into  flasks, 
make  up  to  original  volume  with  distilled  water,  plug  the  flasks 
with  cotton  wool,  and  sterilise  by  methods  B  (2)  or  (3)  (pp.  28, 
29).  This  method  of  neutralisation  is  to  be  recommended  for 
all  ordinary  work. 

In  tliis  medium  the  place  of  the  original  albumins  of  the  meat  is  taken 
by  peptone,  a  soluble  proteid  not  coagulated  by  heat.  Here  it  may  be 
remarked  that  the  commercial  peptone  albumin  is  not  pure  peptone,  but 
a  mixture  of  albmnoses  (see  footnote,  p.  193)  with  a  variable  amount  of 
] m re  peptone.  Tin-  addition  of  the  sodium  chloride  is  necessitated  by 
the  fact  that  alkalinisation  precipitates  some  of  the  phosphates  and 
carbon.it  es  present.  Experience  has  shown  that  sodium  chloride  can 
quite  well  be  substituted.  The  reason  for  the  alkaliuisation  is  that  it  is 
found  that  most  bacteria  grow  best  on  a  medium  slightly  alkaline  to 
litmus.  Some,  e.g.  the  cholera  vibrio,  will  not  grow  at  all  on  even  a 
slightly  acid  medium. 

Standardisation  of  Reaction  of  Media. — While  the  above 
procedure  of  dealing  with  the  reaction  of  a  medium  is  sufficient 
for  ordinary  work,  it  has  been  thought  advisable  to  have  a  more 

1  Some  workers,  instead  of  meat  extract  as  made  above,  use  Liebig's 
extract  of  beef,  2  grammes  to  the  litre. 


34      METHODS  OF  CULTIVATION  OF  BACTERIA 

exact  method  for  making  media  to  be  used  in  growing  organisms, 
the  growth  characteristics  of  which  are  to  be  described  for 
systematic  purposes.  Such  a  method  should  also  be  used  in 
studying  the  changes  in  reaction  produced  in  a  medium  by  the 
growth  of  bacteria.  It,  however,  involves  considerable  difficulty, 
and  should  not  be  undertaken  by  the  beginner.  It  entails  the 
preparation  of  solutions  of  acid  and  alkali  which  may  be  used 
for  determining  the  original  reaction  of  the  medium,  and  for 
accurately  making  it  of  a  definite  degree  of  alkalinity.  Normal l 
and  decinormal  solutions  of  sodium  hydrate  and  hydrochloric 
acid  are  used. 

Preparation  of  Standard  Solutions. — The  first  requisites  here  are 
normal  solutions  of  acid  and  alkali.  The  latter  is  prepared  as  follows  : 
85  grammes  of  pure  sodium  bicarbonate  are  heated  to  dull  redness  for 
ten  minutes  in  a  platinum  vessel  and  allowed  to  cool  in  an  exsiccator  ; 
just  over  54  grammes  of  sodium  carbonate  should  now  be  present.  Any 
excess  is  quickly  removed,  and  the  rest  being  dissolved  in  one  litre  of 
distilled  water,  a  normal  solution  is  obtained.  A  measured  quantity  is 
placed  in  a  porcelain  dish,  and  a  few  drops  of  a  '5  per  cent,  solution  of 
phenol-phthaleine  in  neutral  methylated  spirit  is  added  to  act  as 
indicator.  The  alkali  produces  in  the  latter  a  brilliant  rose-pink,  which, 
however,  disappears  on  the  least  excess  of  acid  being  present.  The 
mixture  is  boiled  and  a  solution  of  hydrochloric  acid  of  unknown  strength 
is  run  into  the  dish  from  a  burette  till  the  colour  goes  and  does  not 
return  after  very  thorough  stirring.  The  strength  of  the  acid  can  then 
be  calculated,  and  a  normal  solution  can  be  obtained.  From  these  two 
solutions  any  strength  of  acid  or  alkali  (such  as  the  decinormal  solution 
of  NaOH  mentioned  below)  may  be  derived. 

As  Eyre  has  suggested,  the  reaction  of  a  medium  may  be 
conveniently  expressed  by  the  sign  +  or  —  to  indicate  acid  or 
alkaline  respectively,  and  a  number  to  indicate  the  number  of 
cubic  centimetres  of  normal  acid  or  alkaline  solution  necessary 
to  make  a  litre  of  the  medium  neutral  to  phenol-phthaleine. 
Thus,  for  example,  "reaction  =  -15,"  will  mean  that  the 
medium  is  alkaline,  and  requires  15  c.c.  of  normal  HC1  to  make 
a  litre  neutral.  It  has  been  found  that  when  a  medium  such 
as  bouillon  reacts  neutral  to  litmus,  its  reaction  to  phenol- 

*A  "normal"  solution  of  any  salt  is  prepared  by  dissolving  an 
' '  equivalent "  weight  in  grammes  of  that  salt  in  a  litre  of  distilled 
water.  If  the  metal  of  the  salt  be  monovalent,  i.e.  if  it  be  replaceable  in 
a  compound  by  one  atom  of  hydrogen  (e.g.  sodium),  an  equivalent  is  the 
molecular  weight  in  grammes.  In  the  case  of  NaCl,  it  would  be  58*5 
grammes  (atomic  Aveight  of  Na  =  23,  of  Cl  =  35'5).  If  the  metal  be 
bivalent,  i.e.  requiring  two  atoms  of  H  for  its  replacement  in  a  compound 
(e.g.  calcium),  an  equivalent  is  the  molecular  weight  in  grammes  divided 
by  two.  Thus  in  the  case  of  CaCl2  an  equivalent  would  be  55  '5  grammes 
(atomic  weight  of  Ca  =  40,  of  C12  =  71 ). 


STANDARDISING  THE  REACTION  OF  MEDIA     35 

I'luhaleine,  according  to  the  above  standard,  is  on  the  average 
+  25.  Now,  as  litmus  was  originally  introduced  by  Koch,  and 
as  nearly  all  bacterial  research  has  been  done  with  media  tested 
by  litmus,  it  is  evidently  difficult  to  say  exactly  what  precise 
degree  of  alkalinity  is  the  optimum  for  bacterial  growth.  It  is 
probably  safe  to  say,  however,  that  when  a  medium  has  been 
rendered  neutral  to  phenol-phthaleine  by  the  addition  of  NaOH, 
the  optimum  degree  is  generally  attained  by  the  addition  of 
from  10  to  15  c.c.  of  normal  HC1  per  litre,  i.e.  the  optimum 
reaction  is  from  +10  to  +15.  In  other  words,  the  optimum 
reaction  for  bacterial  growth  lies,  as  Fuller  has  pointed  out, 
about  midway  between  the  neutral  point  indicated  by  phenol- 
plithaleine  and  the  neutral  point  indicated  by  litmus. 

The  only  objection  to  the  use  of  phenol-phthaleine  is  that 
its  action  is  somewhat  vitiated  if  free  CO2  be  present.  This 
can  be  obviated  by  boiling  any 'medium,  before  it  is  tested, 
in  the  porcelain  dish  into  which  titration  takes  place.  The  soda 
solutions  are  best  stored  in  bottles  such  as  that  shown  in  Fig.  42, 
1  laving  on  the  air  inlet  a  little  bottle  filled  with  soda  lime  and 
fitted  with  tubes  as  in  the  large  one.  The  CO.2  of  the  air  which 
passes  through  is  thus  removed. 

Method. — The  following  procedure  includes  most  of  the 
improvements  introduced  by  Eyre.  The  medium  with  all  its 
constituents  dissolved  is  filtered  and  then  heated  for  about  forty- 
five  minutes  in  the  steamer,  the  maximum  acidity  being  reached 
after  this  time.  Of  the  warm  medium  take  25  c.c.  and  put  in 
a  porcelain  dish,  add  25  c.c.  distilled  water,  and  1  c.c.  phenol- 
phthaleine  solution.  Run  in  decinormal  soda  till  neutral  point 
is  reached,  indicated  by  the  first  trace  of  pink  colour,  the 
mixture  being  kept  hot.1  Repeat  process  thrice,  and  take  the 
mean;  this  divided  by  10  will  give  the  amount  (x)  of  normal 
soda  required  to  neutralise  25  c.c.  of  medium ;  then  40  x  = 
amount  necessary  to  neutralise  a  litre  ;  and  40  x  —  10  =  amount 
of  normal  soda  necessary  to  give  a  litre  its  optimum  reaction. 
Then  measure  the  amount  of  medium  to  be  dealt  with,  and  add 
the  requisite  amount  of  soda  solution. 

Eyre  uses  a  soda  solution  of  ten  times  normal  strength,  which 
is  delivered  out  of  a  1  c.c.  pipette  divided  into  hundred ths ;  this 

1  The  beginner  may  find  considerable  difficulty  in  recognising  the  first 
tint  of  pink  in  the  yellow  bouillon.  A  good  way  of  getting  over  this  is 
to  take  two  samples  of  the  medium,  adding  the  indicator  to  one  only  ; 
then  to  run  the  soda  into  these  from  separate  burettes ;  for  each  few 
drnjis  run  into  the  medium  containing  the  indicator  the  same  amount  is 
run  into  the  other.  Thus  the  recognition  of  the  first  permanent  change 
in  tint  will  be  at  once  recognised  by  comparing  the  two  lots  of  solution. 


36      METHODS  OF  CULTIVATION  OF  BACTERIA 


obviates,  to  a  large  extent,  the  error  introduced  by  increasing 
the  bulk  of  the  medium  if  a  weaker  neutralising  solution  be 
used. 

1  (b).  Glucose  Broth. — -To  the  other  constituents  of  1  (a) 
there  is  added  1  or  2  per  cent,  of  grape  sugar.  The  steps  in  the 
preparation  are  the  same.  Glucose  being  a  reducing  agent,  no 
free  oxygen  can  exist  in  a  medium  containing  it,  and  therefore 
glucose  broth  is  used  as  a  culture  fluid  for  anaerobic  organisms. 

1  (c).  Glycerin  Broth. — The  initial  steps  are  the  same  as  in 
1  (a),  but  after  filtration  6  to  8  per  cent,  of  glycerin  (sp.  grav. 
1'25)  is  added.     This  medium  is  especially  used  for  growing  the 
tubercle  bacillus  when  the  products  of  the  growth  of  the  latter 
are  required. 

2.  Gelatin  Media. — These  are  simply  the  above  broths,  with 
gelatin  added  as  a  solidifying  body. 

2  (a).  Peptone  Gelatin  : — 


Meat  extract 
Sodium  chloride    . 
Peptone  albumin  . 
Gelatin 


.      1000  c.c. 

5  grms. 
10     „ 
100-150 


(The  "gold  label "  gelatin  of  Coignet  et  Cie,  Paris,  is  the  best.) 
The  gelatin  is  cut  into  small  pieces,  and  added  with  the  other 
constituents  to  the  extract ;  they  are  then  thoroughly  melted  on 
a  sand  bath,  or  in  the  "  Koch."  The 
fluid  medium  is  then  rendered  slightly 
alkaline,  as  in  1  (a),  and  filtered 
through  filter  paper.  As  the  medium 
must  not  be  allowed  to  solidify 
during  the  process,  it  must  be  kept 
warm.  This  is  effected  by  putting 
the  flask  and  funnel  into  a  tall 
Koch's  steriliser,  in  which  case  the 
funnel  must  be  supported  on  a 
tripod  or  diaphragm,  as  there  is  great 
danger  of  the  neck  of  the  flask  break- 
ing if  it  has  to  support  the  funnel 
and  its  contents.  The  filtration  may 
also  be  carried  out  in  a  funnel 
with  water-jacket  which  is  heated,  as 

shown  in  Fig.  7.  Whichever  instrument  be  used,  before  filtering 
shake  up  the  melted  medium,  as  it  is  apt  while  melting  to  have 
settled  into  layers  of  different  density.  Sometimes  what  first 
comes  through  is  turbid.  If  so,  replace  it  in  the  unfiltered 


FIG.  7. — Hot-water  funnel. 


AGAR  MEDIA  37 

part :  often  the  subsequent  filtrate  in  such  circumstances  is 
quite  clear.  A  litre  flask  of  the  finished  product  ought  to  be 
quite  transparent.  If,  however,  it  is  partially  opaque,  add 
the  white  of  an  egg,  shake  up  well,  and  boil  thoroughly  over 
the  sand  bath.  The  consequent  coagulation  of  the  album  in 
carries  down  the  opalescent  material,  and,  on  making  up  with 
distilled  water  to  the  original  quantity  and  refiltering,  it  will  be 
found  to  be  clear.  The  flask  containing  it  is  then  plugged  with 
cotton  wool  and  sterilised,  best  by  method  B  (2),  p.  28.  If  the 
autoclave  be  used  the  temperature  employed  must  not  be  above 
105°  C.,  and  exposure  not  more  than  a  quarter  of  an  hour  on 
three  successive  days.  Too  much  boiling,  or  boiling  at  too  high 
a  temperature,  as  has  been  said,  causes  a  gelatin  medium  to  lose 
its  property  of  solidification.  The  exact  percentage  of  gelatin 
used  in  its  preparation  depends  on  the  temperature  at  which 
growth  is  to  take  place.  Its  firmness  is  its  most  valuable 
characteristic,  and  to  maintain  this  in  hot  summer  weather,  15 
parts  per  100  are  necessary.  A  limit  is  placed  on  higher  per- 
centages by  the  fact  that,  if  the  gelatin  be  too  stiff,  it  will  split 
on  the  perforation  of  its  substance  by  the  platinum  needle  used 
in  inoculating  it  with  a  bacterial  growth ;  1 5  per  cent,  gelatin 
melts  at  about  24°  C.  For  ordinary  use  in  British  laboratories 
10  per  cent,  gelatin  is  a  sufficient  strength. 

2  (b).  Glucose   Gelatin. — The  constituents  are  the  same  as 
2  (a),  with  the  addition  of  1  to  2  per  cent,  of  grape  sugar.     The 
method  of  preparation  is  identical.     This  medium  is  used  for 
growing  anaerobic  organisms  at  the  ordinary  temperatures. 

3.  Agar  Media  (French,  "  ge'lose ").— The  disadvantage  of 
gelatin  is  that  at  the  blood  temperature  (38°  C.),  at  which  most 
pathogenic  organisms  grow  best,  it  is  liquid.  To  get  a  medium 
which  will  be  solid  at  this  temperature,  agar  is  used  as  the 
stiffening  agent  instead  of  gelatin.  Unlike  the  latter,  which 
is  a  proteid,  agar  is  a  carbohydrate.  It  is  derived  from  the 
stems  of  various  seaweeds  growing  in  the  Chinese  seas,  com- 
mercially classed  together  as  "  Ceylon  Moss."  For  bacteriological 
purposes  the  dried  stems  of  the  seaweed  may  be  used,  but  there 
is  in  the  market  a  purified  product  in  the  form  of  a  powder, 
which  is  preferable. 

3  (a).  "  Ordinary  "  Agar. — This  has  the  following  composi- 
tion : — 

Mi-at  extract 1000  c.c. 

Sodium  chloride         ....  5  grms 

Peptone  albumin       .         .         .         .          10     „ 
ir  15 


38     METHODS  OF  CULTIVATION  OF  BACTERIA 

Cut  up  the  agar  into  very  fine  fragments  (in  fact  till  it  is  as 
nearly  as  possible  dust),  add  to  the  meat  extract  with  the  other 
ingredients,  and  preferably  allow  to  stand  all  night.  Then  boil 
gently  in  a  water  bath  for  two  or  three  hours,  till  the  agar  is 
thoroughly  melted.  The  process  of  melting  may  be  hastened 
by  boiling  the  medium  in  a  sand  bath  and  passing  through  it  a 
stream  of  steam  generated  in  another  flask ;  the  steam  is  led 
from  the  second  flask  by  a  bent  glass  tube  passing  from  just 
beneath  the  cork  to  beneath  the  surface  of  the  medium  (Eyre). 
After  melting,  render  slightly  alkaline  with  sodium  hydrate 
solution,  make  up  to  original  volume  with  distilled  water,  and 
filter.  Filtration  here  is  a  very  slow  process,  and  is  best  carried 
out  in  a  tall  Koch's  steriliser.  In  doing  this,  it  is  well  to  put  a 
glass  plate  over  the  filter  funnel  to  prevent  condensation  water 
from  dropping  oft7  the  lid  of  the  steriliser  into  the  medium.  If 
a  slight  degree  of  turbidity  may  be  tolerated,  it  is  sufficient  to 
filter  through  a  felt  bag  or  jelly  strainer.  Plug  the  flask  con- 
taining the  filtrate,  and  sterilise  either  in  autoclave  for  fifteen 
minutes  or  in  Koch's  steriliser  for  one  and  a  half  hours. 
Agar  melts  just  below  100°  C.,  and  on  cooling  solidifies  about 
39°  C. 

3  (b).  Glycerin  Agar.— To  3  (a)  after  filtration  add  6  to  8 
per  cent,  of  glycerin  and  sterilise  as  above.  This  is  used 
especially  for  growing  the  tubercle  bacillus. 

3  (c).  Glucose  Agar. — Prepare  as  in  3  (a),  but  add  1  to  2 
per  cent,  of  grape  sugar  along  with  agar.  This  medium  is  used 
for  the  culture  of  anaerobic  organisms  at  temperatures  above  the 
melting-point  of  gelatin.  It  is  also  an  excellent  culture  medium 
for  some  aerobes,  e.g.  the  b.  diphtherise. 

These  bouillon,  gelatin,  and  agar  preparations  constitute 
the  most  frequently  used  media.  Growths  in  bouillon  do  not 
usually  show  any  characteristic  appearances  which  facilitate 
classification,  but  such  a  medium  is  of  great  use  in  investigating 
the  soluble  toxic  products  of  bacteria.  The  most  characteristic 
developments  of  organisms  take  place  on  the  gelatin  media. 
These  have,  however,  the  disadvantage  of  not  being  available 
when  growth  is  to  take  place  at  any  temperature  above  24°  C. 
For  higher  temperatures  agar  must  be  employed.  Agar  is,  how- 
ever, never  so  transparent.  Though  quite  clear  when  fluid,  on 
solidifying  it  always  becomes  slightly  opaque.  Further,  growths 
upon  it  are  never  so  characteristic  as  those  on  gelatin.  It  is, 
for  instance,  never  liquefied,  whereas  some  organisms,  by  their 
growth,  liquefy  gelatin  and  others  do  not — a  fact  of  prime 
importance. 


SPECIAL  CULTURE  MEDIA  39 

SPECIAL  CULTURE  MEDIA. 

An  enormous  variety  of  different  media  has  been  brought 
forward  for  use  in  cases  either  where  special  difficulty  is  ex- 
perienced in  getting  an  organism  to  grow,  or  where  some  special 
growth  characteristic  is  to  be  studied.  It  is  impossible  to  do 
more  than  give  the  chief  of  these. 

Peptone  Solution. 

A  simple  solution  of  peptone  (Witte)  constitutes  a  suitable 
culture  medium  for  many  bacteria.  The  peptone  in  the  propor- 
tion of  1  to  2  per  cent.,  along  with  '5  per  cent.  NaCl,  is  dissolved 
in  distilled  water  by  heating.  The  fluid  is  then  filtered,  placed 
in  tubes,  and  sterilised.  The  reaction  is  usually  distinctly 
alkaline,  which  condition  is  suitable  for  most  purposes.  For 
special  purposes  the  reaction  may  be  standardised.  In  such  a 
solution  the  cholera  vibrio  grows  with  remarkable  rapidity.  It 
is  also  much  used  for  testing  the  formation  of  indol  by  a 
particular  bacterium ;  and  by  the  addition  of  one  of  the  sugars 
to  it  the  fermentative  powers  of  an  organism  may  be  tested 
(p.  80).  Litmus  may  be  added  to  show  any  change  in  reaction. 

Media  containing  an  Indicator. 

Litmus  Media. — To  any  of  the  ordinary  media  litmus  (French, 
tournesol)  may  be  added  to  show  change  in  reaction  during 
bacterial  growth.  The  litmus  is  added,  before  sterilisation,  as 
a  strong  watery  solution  (e.g.  the  Kubel-Tiemann  solution,  vide 
p.  48)  in  sufficient  quantity  to  give  the  medium  a  distinctly 
bluish  tint.  During  the  development  of  an  acid  reaction  the 
colour  changes  to  a  pink,  and  may  subsequently  be  dis- 
charged. 

Neutral  Red  Media. — This  dye  has  been  introduced  as  an  aid 
in  determining  the  presence  or  absence  of  members  of  the  b.  coli 
group,  especially  in  the  examination  of  water.  The  media  found 
most  suitable  are  agar  or  bouillon  containing  '5  per  cent,  of 
lactose,  to  which  '5  per  cent,  of  a  1  per  cent,  watery  solution 
of  neutral  red  is  added.  The  alkaline  medium  is  of  "a  yellowish 
brown  colour  which  on  the  presence  of  acid  passes  into  a  deep 
rose  red.  Sometimes  there  subsequently  occurs  a  change  to  a 
fluorescent  green,  caused  apparently  by  a  change  in  the  com- 
position of  the  dye,  as  the  fluorescence  is  not  discharged  by 
addition  of  alkali. 


40     METHODS  OF  CULTIVATION  OF  BACTERIA 

Blood  Serum  Media. 

Koch's  Blood  Serum. — Koch  introduced  this  medium,  and  it 
is  prepared  as  follows  :  Plug  the  mouth  of  a  tall  cylindrical  glass 
vessel  (say  of  1000  c.c.  capacity)  with  cotton  wool,  and  sterilise 
by  steaming  it  in  a  Koch's  steriliser  for  one  and  a  half  hours. 
Take  it  to  the  place  where  a  horse,  ox,  or  sheep  is  to  be  killed. 
When  the  artery  or  vein  of  the  animal  is  opened,  allow  the  first 
blood  which  flows,  and  which  may  be  contaminated  from  the 
hair,  etc.,  to  escape;  fill  the  vessel  with  the  blood  subsequently 
shed.  Carry  carefully  back  to  the  laboratory  without  shaking, 
and  place  for  twenty-four  hours  in  a  cool  place,  preferably  an  ice 
chest.  The  clear  serum  will  separate  from  the  clotted  blood. 
If  a  centrifuge  is  available,  a  large  yield  of  serum  may  be  obtained 
by  centrifugalising  the  freshly  drawn  blood.  If  coagulation  has 
occurred,  the  clot  must  first  be  thoroughly  broken  up.  With  a 
sterile  10  c.c.  pipette,  transfer  this  quantity  of  serum  to  each  of 
a  series  of  test-tubes  which  must  previously  have  been  sterilised 
by  dry  heat.  The  serum  may,  with  all  precautions,  have  been 
contaminated  during,  the  manipulations,  and  must  be  sterilised. 
As  it  will  coagulate  if  heated  above  68°  C.,  advantage  must  be 
taken  of  the  intermittent  process  of  sterilisation  at  57°  C. 
[method  B  (4)].  It  is  therefore  kept  for  one  hour  at  this 
temperature  on  each  of  eight  successive  days.  It  is  always 
well  to  incubate  it  for  a  day  at  37°  C.  before  use,  to  see  that 
the  result  is  successful.  After  sterilisation  it  is  "inspissated," 
by  which  process  a  clear  solid  medium  is  obtained.  "  Inspissa- 
tion  "  is  probably  an  initial  stage  of  coagulation,  and  is  effected 
by  keeping  the  serum  at  65°  C.  till  it  stiffens.  This  temperature 
is  just  below  the  coagulation  point  of  the  serum.  The  more 
slowly  the  operation  is  performed  the  clearer  will  be  the  serum. 
The  apparatus  used  for  the  purpose  is  one  of  the  various  forms 
of  serum  steriliser  (e.g.  Fig.  8),  generally  a  chamber  with  water- 
jacket  heated  with  a  Bunsen  below.  The  temperature  is  con- 
trolled by  a  gas  regulator,  and  such  an  apparatus  can,  by  altering 
the  temperature,  be  used  either  for  sterilisation  or  inspissation. 
As  is  evident,  the  preparation  of  this  medium  is  tedious,  but  its 
use  is  necessary  for  the  observation  of  particular  characteristics 
in  several  pathogenic  bacteria,  notably  the  tubercle  bacillus. 
Pleuritic  and  other  effusions  may  be  prepared  in  the  same  way, 
and  used  as  media,  but  care  must  be  taken  in  their  use,  as  we 
have  no  right  to  say  that  pathological  effusions  have  the  same 
chemical  composition  as  normal  serum. 

If    blood    be    collected  with  strict   aseptic    precautions,  then 


BLOOD  SERUM  MEDIA 


41 


sterilisation  of  the  serum  is  unnecessary.     To  this  end  the  mouth 

of  the   cylinder  used  for  collecting  the  blood,  instead  of  being 

plugged    with    wool,    has  an    indiarubber  bung  inserted    in    it 

through   which  two  bent 

glass    tubes    pass.       The 

outer  end  of  one  of  these 

is    of    convenient    length, 

and,  before  sterilisation,  a 

large  cap  of  cotton   wool 

is  tied  over  it ;  the  other 

tube   is    plugged   with   a 

piece  of  cotton  wool.     In 

the    slaughter  -  house    the 

cap  is  removed   and    the 

tube  is  inserted  into  the 

blood-vessel  as  a  cannula. 

The  cylinder  is  thus  easily 

filled.    Another  method  is 

to    conduct  the   blood    to 

the     cylinder    by    means 

of    a     sterilised     cannula 

and  indiarubber  tube,  the 

former  being   inserted   in 

the  blood-vessel.    In  every 

case  the  serum   must   be 

incubated   before   use,   to 

make     sure     that     it    is 

sterile. 

Coagulated  Blood 
Serum. — If  fresh  serum 
be  placed  in  sterile  tubes 
and  be  steamed  in  the 
sloped  position  for  an 
hour,  it  coagulates,  and 


FIG.  8. — Blood  serum  inspissator. 


there  is  thus  obtained  a  solid  medium  very  useful  for  the  growth 
of  the  diphtheria  bacillus  for  diagnostic  purposes. 

Loffler's  Blood  Serum. — This  is  the  best  medium  for  the 
growth  of  tin-  b.  diphtheria?,  and  may  be  used  for  other  organisms. 
It  has  the  following  composition  :  Three  parts  of  calf's  or  lamb's 
blood  serum  are  mixed  with  one  part  ordinary  neutral  peptone 
bouillon  nijulo  from  veal  with  1  }>er  cent,  of  grape  sugar  added 
to  it.  Though  this  is  the  original  formula,  it  can  be  made  from 
ox  or  -Invp  scrum  and  beef  bouillon  without  its  qualities  being 
markedly  impaired.  Sterilise  by  method  B  (4)  as  above  (p.  30). 


42      METHODS  OF  CULTIVATION  OF  BACTEEIA 

Alkaline  Blood  Serum  (Lorrain  Smith's  Method). — To  each 
100  c.c.  of  the  serum  obtained  as  before,  add  1  to  1*5  c.c.  of  a 
10  per  cent,  solution  of  sodium  hydrate  and  shake  gently.  Put 
sufficient  of  the  mixture  into  each  of  a  series  of  test-tubes,  and, 
laying  them  on  their  sides,  sterilise  by  method  B  (2).  If  the 
process  of  sterilisation  be  carried  out  too  quickly,  bubbles  of  gas 
are  apt  to  form  before  the  serum  is  solid,  and  these  interfere  with 
the  usefulness  of  the  medium.  Dr.  Lorrain  Smith  informs  us 
that  this  can  be  obviated  if  the  serum  be  solidified  high  up  in 
the  Koch's  steriliser,  in  which  the  water  is  allowed  only  to 
simmer.  In  this  case  sterilisation  ought  to  go  on  for  one  and 
a  half  hours.  A  clear  solid  medium  (consisting  practically  of 
alkali-albumin)  is  thus  obtained,  and  he  has  found  it  of  value 
for  the  growth  of  the  organisms  for  which  Koch's  serum  is  used, 
and  especially  for  the  growth  of  the  b.  diphtheriae.  Its  great 
advantage  is  that  aseptic  precautions  in  obtaining  blood  from  the 
animal  are  not  necessary,  and  it  is  easily  sterilised. 

Marmorek's  Serum  Media. — There  has  always  been  a  diffi- 
culty in  maintaining  the  virulence  of  cultures  of  the  pyogenic 
streptococci,  but  Marmorek  has  succeeded  in  doing  so  by  growing 
them  on  the  following  media,  which  are  arranged  in  the  order  of 
their  utility  : — 

1.  Human  serum  2  parts,  bouillon  1  part. 

2.  Pleuritic  or  ascitic  serum  1  part,  bouillon  2  parts. 

3.  Asses'  or  mules'  serum  2  parts,  bouillon  1  part. 

4.  Horse  serum  2  parts,  bouillon  1  part. 

Human  serum  can  be  obtained  from  the  blood  shed  in 
venesection,  the  usual  aseptic  precautions  being  taken.  In  the 
case  of  these  media,  sterilisation  is  effected  by  method  B  (4),  and 
they  are  used  fluid. 

Serum  Media  for  Gonococcus. — The  two  following  media 
will  be  found  suitable.  Wertheim's  medium  consists  of  one  part 
of  sterile  human  serum  (conveniently  obtained  from  placental 
blood)  and  two  parts  of  agar.  The  agar  is  sterilised,  and  fluid 
is  allowed  to  cool  to  40°  C. ;  the  serum  is  then  added,  and  the 
mixture  is  allowed  to  solidify  in  the  sloped  position. 

Gurd's  medium  is  a  2  per  cent,  agar  with  acid  reaction  +  6 
to  phenol-phthaleine  (p.  34),  with  defibrinated  human  blood  added 
in  the  proportion  of  about  5  drops  to  5  c.c.  of  agar  ;  the  blood 
is  added  to  the  melted  agar  as  in  Wertheim's  medium. 
W.  B.  M.  Martin  recommends  the  substitution  of  sodium 
phosphate  ('5  per  cent.)  for  sodium  chloride  in  the  preparation 
of  the  agar,  and  uses  fluid  human  serum  sterilised  at  57°  C.  in 


BLOOD  MEDIA  43 

place  of  blood.  He  also  finds  that  the  same  agar  medium 
allowed  to  solidify  and  then  smeared  on  the  surface  with  a  drop 
or  two  of  human  serum  gives  excellent  results. 

Any  of  these  media  may  be  used  for  plate  cultures,  the  agar 
being  melted  and  cooled  to  40"  C.  as  for  agar  plates  ;  the  serum 
or  blood  is  then  added ;  the  mixture  is  inoculated  in  the  usual 
way  and  poured  out  in  Petri  dishes. 

"Nasgar."—  This  is  a  serum  medium  introduced  by  Gordon  for  the 
isolation  of  the  meningococcus.  It  is  prepared  as  follows  : — 

Ascitic  fluid     .         .         .         .         .         .     15  c.c. 

Distilled  water         .         .         .         .         .     35  c.c. 

Nutrose1 1  gramme. 

Put  in  a  flask,  bring  to  boil,  constantly  shaking  till  ebullition  occurs ; 
filter.  Of  the  resultant  fluid  take  one  part  and  add  two  parts  of  ordinary 
iM.'l'tuiie  agar.  Steam  for  half  an  hour  and  place  in  tubes. 

Blood  Media. 

Blood  -  Smeared  Agar.  —  This  medium  was  introduced  by 
Pfeili'er  tor  growing  the  influenza  bacillus,  and  it  has  been  used 
for  the  organisms  which  are  not  easily  grown  on  the  ordinary 
media,  e.y.  the  gonococcus  and  the  pneumococcus.  Human 
Mood  or  the  blood  of  animals  maybe  used.  "Sloped  tubes" 
(vide  p.  53)  of  agar  are  employed  (glycerin  agar  is  not  so 
suitable).  Purify  a  finger  first  with  1-1000  corrosive  sublimate, 
dry,  and  then  wash  with  absolute  alcohol  to  remove  the  sub- 
limate. Allow  the  alcohol  to  evaporate.  Prick  with  a  needle 
sterilised  by  heat,  and,  catching  a  drop  of  blood  in  the  loop  of  a 
Merile  platinum  wire  (vide  p.  54),  smear  it  on  the  surface  of  the 
agar.  The  excess  of  the  blood  runs  down  and  leaves  a  film 
on  the  surface.  Cover  the  tubes  with  indiarubber  caps,  and 
yicubate  them  for  one  or  two  days  at  38°  C.  before  use,  to 
make  certain  that  they  are  sterile.  Agar  poured  out  in  a  thin 
layer  in  a  Petri  dish  may  be  smeared  with  blood  in  the  same 
\\ay  and  used  for  cultures. 

Serum  Agar  is  prepared  in  a  similar  way  by  smearing  the 
surface  of  the  agar  with  blood  serum,  or  by  adding  a  few  drops 
of  serum  to  the  tube  and  then  allowing  it  to  flow  over  the 
si  ir  face. 

Blood   Agar. — For    many  purposes  (e.g.  the  growth  of   the 

whooping-cough  bacillus,  the  bacillus  of  soft  sore,  the  cultivation 

of  trypanosomes  and  Leishmaniae),  the  use  of  agar  containing 

detibrinated  blood,  especially  rabbit   blood,  is   desirable.     The 

1  Nutrose  is  an  alkaline  preparation  of  casein. 


44     METHODS  OF  CULTIVATION  OF  BACTERIA 

blood  may  be  obtained  in  several  ways,  preferably  by  bleeding 
from  the  carotid.  For  this  purpose  the  vessel  is  exposed  and 
as  long  a  portion  as  possible  is  cleaned.  This  is  ligatured  high 
up,  and  a  ligature  is  loosely  applied  round  the  lower  part  of 
the  vessel  in  such  a  way  as  not  to  constrict  it,  The  vessel  is 
clamped  above  this  ligature,  and  with  scissors  an  oblique 
opening  is  made  in  its  side.  The  clamp  being  removed,  the 
stream  of  blood  is  directed  by  means  of  the  ligature  into  the 
mouth  of  a  stout  sterile  flask,  which  ought  to  contain  some 
fragments  of  broken  glass  rod.  During  the  bleeding  the  flask 
should  be  gently  agitated,  and  when  filled  should  be  shaken  in 
a  bath  of  water  just  below  blood-heat.  We  have  found  that 
sterile  blood  can  be  obtained  from  the  ear  vein  of  the  rabbit  by 
the  method  of  bleeding  to  be  subsequently  described.  The  ear 
is  well  washed  with  lysol,  the  lysol  dried  off  with  sterile  wool, 
absolute  alcohol  dropped  on  and  allowed  to  evaporate,  arid  the 
blood  withdrawn.  The  first  c.c.  or  so  is  rejected. 

However  the  blood  is  obtained,  after  defibrination  it  is  warmed 
to  45°  C.,  and  added  to  agar  of  the  same  temperature  in  the 
proportion  of  about  one-third  of  blood  and  two-thirds  of  agar. 
Needless  to  say,  such  media  must  be  incubated  before  use  to 
ensure  that  bacteria  have  not  gained  access  during  preparation. 

Bordet  and  Gengou's  Medium  for  Bacillus  of  Whooping-cough.— 

An  extract  of  potato  is  first  prepared  by  adding  two  parts  of  water  contain- 
ing 4  per  cent,  of  glycerin  to  one  part  of  potato  chips  ;  the  mixture  is 
then  boiled  and-  the  fluid  is  separated  off.  An  agar  medium  is  then 
prepared  of  the  following  composition  :  potato  extract,  50  c.c.;  '6  per  cent, 
solution  of  sodium  chloride,  150  c.c.;  and  agar,  Sgrrns.  Of  this  medium, 
2-3  c.c.  is  placed  in  each  of  a  series  of  test- tubes,  and  then  to  each  there 
is  added,  by  the  method  described  in  the  preceding  paragraph,  an  equal 
part  of  defibrinated  rabbit's  (or  better,  human)  blood,  obtained  by  aseptic 
precautions.  The  mixture  is  then  allowed  to  solidify  in  the  sloped 
position.  This  medium  is  also  very  suitable  for  the  growth  of  the 
gonococcus,  meningococcus,  and  influenza  bacillus. 

t 

Blood- Alkali- Agar  (Dieudonne). — This  medium,  introduced 
for  the. culture  of  the  cholera  spirillum,  for  which  purpose  it  has 
been  found  extremely  suitable,  has  the  property  of  inhibiting 
the  growth  of  most  of  the  intestinal  bacteria ;  for  example,  the 
b.  coli  does  not  grow  on  it,  or  does  so  very  slightly.  A  blood- 
alkali  solution  is  prepared  by  adding  equal  parts  of  defibrinated 
ox  blood  and  of  normal  caustic  soda  solution ;  the  solution  may 
then  be  sterilised  in  the  steam  steriliser.  Of  this  solution  three 
parts  are  added  to  seven  parts  of  ordinary  peptone-agar  rendered 
neutral  to  litmus,  and  the  mixture  is  disposed  in  test-tubes. 


POTATOES  AS  CULTURE  MATERIAL     45 

Novy  and  MacNeal's  Medium  for  Culture  of  Trypanosomes.  —  1  25 

grammes  rabbit  or  ox  flesh  is  treated  with  1000  c.c.  distilled  water, 
;is  in  making  ordinary  bouillon,  and  there  are  added  to  the  meat 
extract  20  ^rms.  Witt<-'s  peptone,  5  grms.  sodium  chloride,  20  grms.  agar, 
ami  in  O.C,  iiuniial  sodium  carbonate.  The  medium  is  placed  in  tubes 
and  sterilised  in  the  autoclave  at  110°  C.  for  thirty  minutes.  It  is 
cooled  to  50°  C.,  and  there  is  added  to  the  medium  in  each  tube  twice 
its  volume  of  defibrinated  rabbit  blood,  which  has  been  prepared  with 
all  asrptic  precautions  ;  the  tubes  are  allowed  to  set  in  the  inclined 
position.  In  inoculating  such  tubes  they  are  placed  in  an  upright 
position  for  a  few  minutes,  and  then  the  infective  material  is  introduced. 

1'otatoes  as  Culture  Material. 

('/)  In  Potato  Jars.  —  The  jar  consists  of  a  round,  shallow, 

glass  vessel  with  a  similar  cover  (vide  Fig.   9).     It  is  washed 

with    1-1000   corrosive  sublimate, 

and  a  piece  of  circular  filter  paper, 

moistened  with  the  same,  is   laid 

in  its  bottom.     On  this  latter  are 

placed    four    sterile    watch-glasses. 

Two    firm,    healthy,    small,    round 

potatoes    as     free    from    eyes    as 

possible,  and  with  the  skin  whole, 

are    scrubbed    well    with    a   brush 

under  the  tap  arid  steeped  for  two  FIG.  9.—  Potato  jar. 

or  three  hours  in  1-1000  corrosive 

sublimate.'    They  are  steamed  in  the  Koch's  steriliser  for  thirty 

minutes  or  longer,  or  in  the  autoclave  for  a  quarter  of  an  hour. 

When  cold,  each  is  grasped  between  the  left  thumb  and  forefinger 

(which  have  been  sterilised  with  sublimate)  and  cut  through  the 

middle  with  a  sterile  knife.     It  is  best  to  have  the  cover  of  the 

jar  raised  by  an  assistant,  and  to  perform  the  cutting  beneath  it. 

Each  half  is  put  in  one  of  the  watch-glasses,  the  cut  surfaces, 
which  are  then  ready  for  inoculation 
with  a  bacterial  growth,  being  upper- 
most. Smaller  jars,  each  of  which 
holds  half  of  a  potato,  are  also  used 
in  the  same  way  and  are  very  con- 


FIG.  10.  -Cylinder  of  potato  « 

cut  obliquely.  (6)    By    Slices    in    Tubes.  —  This 

method,  introduced  by  Ehrlich,  is  the 

best  means  of  utilising  potatoes  as  a  medium.  A  large,  long 
potato  is  well  washed  and  scrubbed,  and  peeled  with  a  clean 
knife.  A  cylinder  is  then  bored  from  its  interior  with  an  apple 
corer  or  a  large  cork  borer,  and  is  cut  obliquely,  as  in  Fig  10. 


46      METHODS  OF  CULTIVATION  OF  BACTERIA 


Two  wedges  are  thus  obtained,  each  of  which  is  placed  broad 
end  downward  in  a  test-tube  of  special  form  (see  Fig.  11).  In 
the  wide  part  at  the  bottom  of  this  tube  is  placed  a  piece  of 
cotton  wool,  which  catches  any  condensation  water 
which  may  form.  The  wedge  rests  on  the  con- 
striction above  this  bulbous  portion.  The  tubes, 
washed,  dried,  and  with  cotton  wool  in  the  bottom 
and  in  the  mouth,  are  sterilised  before  the  slices  of 
potato  are  introduced.  After  the  latter  are  in- 
serted, the  tubes  are  sterilised  in  the  Koch  steam 
steriliser  for  one  hour,  or  in  the  autoclave  for 
fifteen  minutes,  at  115°  C.  An  ordinary  test-tube 
may  be  used  with  a  piece  of  sterile  absorbent  wool 
in  its  bottom,  on  which  the  potato  may  rest. 

Glycerin  potato,  suitable  for  the  growth  of  the 
tubercle  bacillus,  may  be  prepared  by  covering  the 
slices  in  the  tubes  with  6  per  cent,  solution  of 
glycerin  in  water,  and  steaming  for  half  an  hour. 
The  fluid  is  then  poured  off  and  the  sterilisation 
continued  for  another  half-hour. 
FIG.  11.—  Potatoes  ought  not  to  be  prepared  long  before 

Ehrlich's   being  used,  as  the  surface  is  apt  to   become  dry 
taini'ng  piece   an(^  discoloured.     It  is  well  to  take  the  reaction  of 
of  potato.        the  potato  with  litmus  before  sterilisation,  as  this 
varies  ;  normally  in  young  potatoes  it  is  weakly  acid. 
The  reaction  of  the  potato  may  be  more  accurately  estimated  by 
steaming  the  potato  slices  for  a  quarter  of  an  hour  in  a  known 
quantity  of  distilled  water,  and  then  estimating  the  reaction  of 
the  water  by  phenol-phthaleine.     The  required  degree  of  acidity 
or  alkalinity  is  obtained  by  adding  the   necessary  quantity  of 
HC1  or  NaOH  solution  (p.  35),  and  steaming  for  other  fifteen 
minutes.     The  water  is  then   poured  off  and  sterilisation  con- 
tinued for  another  half-hour.     Potatoes  before  being  inoculated 
ought  always  to  be  incubated  at  37°  C.  for  a  night,  to  make 
sure  that  their  sterilisation  has  been  successful. 


Milk  as  a  Culture  Medium. 

This  is  a  convenient  medium  for  observing  the  effects  of 
bacterial  growth  in  changing  the  reaction,  in  coagulating  the 
soluble  albumin,  and  in  fermenting  the  lactose.  It  is  prepared 
as  follows  :  Fresh  milk  is  taken,  preferably  after  having  had  the 
cream  "  separated  "  by  centrifugalisation,  as  is  practised  in  the 
best  dairies,  and  is  steamed  for  fifteen  minutes  in  the  Koch ;  it 


MEDIA  FOR  SEPARATING  BACTERIAL  GROUPS     47 

is  then  set  aside  in  an  ice  chest  or  cool  place  over  night  to 
facilitate  further  separation  of  cream.  The  milk  is  siphoned  off 
from  beneath  the  cream.  The  reaction  of  fresh  milk  is  alkaline. 
If  great  accuracy  is  necessary,  any  required  degree  of  reaction 
may  be  obtained  by  the  titration  methoc|.  It  is  then  placed  in 
tubes,  and  sterilised  by  methods  B  (2)  or  B  (3). 

Bread  Paste. 

This  is  useful  for  growing  torulse,  moulds,  etc.  Some 
ordinary  bread  is  cut  into  slices,  and  then  dried  in  an  oven  till 
it  is  so  dry  that  it  can  be  pounded  to  a  fine  powder  in  a  mortar, 
or  rubbed  down  with  the  fingers  and  passed  through  a  sieve. 
Some  100  c.c.  flasks  are  washed,  dried,  and  sterilised,  and  a 
layer  of  the  powder  half  an  inch  thick  placed  on  the  bottom. 
Distilled  water,  sufficient  to  cover  the  whole  of  it,  is  then  run  in 
with  a  pipette  held  close  to  the  surface  of  the  bread,  and,  the 
cotton-wool  plugs  being  replaced,  the  flasks  are  sterilised  in  the 
Koch's  steriliser  by  method  B  (2).  The  reaction  is  slightly 
acid. 

Jfedia  used  for  separating  the  Members  of  Jlacterial  Groups. 

A  great  number  of  media  have  been  devised  for  use  in 
differentiating  the  members  of  the  coli-typhoid  and  other 
bacterial  groups.  The  general  feature  of  these  media  is  that 
they  contain  certain  substances,  often  sugars,  which  tend  to 
bring  out  the  special  characters  of  the  organism  under  investiga- 
tion. Sometimes  also  substances  are  present  which  inhibit  the 
growth  of  bacteria  other  than  those  belonging  to  the  group. 
The  following  are  the  media  which  here  deserve  most  attention  : — 

Hiss's  Serum  Water  Media.— These  are  composed  of  one  part  of  ox's 
serum  and  three  parts  of  distilled  water  with  1  per  cent,  litmus  ;  various 
sugars  in  a  pure  condition  are  added  in  the  proportion  of  1  per  cent. 
The  development  of  acid  by  fermentation  is  shown  by  the  alteration  of 
the  colour  and  by  coagulation  of  the  medium.  These  media  do  not 
coagulate  at  100°  C.,  and  thus  can  be  sterilised  in  the  steam  steriliser. 
They  have  been  extensively  used  by  American  workers  in  studying  the 
fermentative  properties  of  the  b.  dysenteriae,  b.  coli,  etc. 

Drigalski  and  Conradi's  Medium. — This  is  one  of  the  media  used  for 
tin-  study  of  intestinal  bacteria,  and  especially  for  the  isolation  of  the 
typhoid  group  of  organisms,  (a)  Three  pounds  of  meat  are  treated  with 
two  litres  of  water  overnight ;  the  fluid  is  separated  as  usual,  boiled  for 
an  hour,  filtered,  and  there  are  added  20  grammes  Witte's  peptone,  20 
grammes  nutrose,  10  grammes  sodium  chloride  ;  the  mixture  is  then 
boiled  for  an  hour,  60  grammes  finest  agar  are  added,  and  it  is  placed  in 


48      METHODS  OF  CULTIVATION  OF  BACTERIA 

the  autoclave  till  melted  (usually  one  hour) ;  it  is  then  rendered  slightly 
alkaline  to  litmus,  filtered,  and  boiled  for  half  an  hour,  (b)  260  c.c. 
Kubel-Tiemann  litmus1  solution  is  boiled  for  ten  minutes,  30  grammes 
milk  sugar  (chemically  pure)  are  added,  and  the  mixture  is  boiled  for 
fifteen  minutes  ;  (a)  and  (b)  are  then  mixed  hot,  well  shaken,  and,  if 
necessary,  the  slightly  alkaline  reaction  restored.  There  are  then  added 
4  c.c.  of  a  10  per  cent,  sterile  solution  of  water- free  sodium  carbonate  and 
20  c.c.  of  a  freshly  prepared  solution  made  by  dissolving  °1  gramme 
crystal-violet  B,  Hoechst,  in  100  c.c.  hot  sterile  distilled  water.  This 
is  the  finished  medium,  and  great  care  must  be  taken  not  to  overheat  it 
or  to  heat  it  too  long,  as  changes  in  the  lactose  may  be  originated.  It 
is  convenient  to  distribute  the  medium  in  80  c.c.  flasks. 

The  principle  of  the  medium  is  that  while  there  is  a  food  supply  very, 
favourable  to  the  b.  typhosus  and  the  b.  coli,  the  antiseptic  action  of  the 
crystal- violet  tends  to  inhibit  the  growth  of  other  bacteria  likely  to 
occur  in  material  which  has  been  subjected  to  intestinal  contamination. 
In  examining  fteces,  a  little  is  rubbed  up  in  from  ten  to  twenty  times  its 
volume  of  sterile  bouillon  ;  in  the  case  of  urine  or  water,  the  fluid  is 
centrifugalised  and  the  deposit  or  lower  portion  is  used  for  the  inocula- 
tion procedures. 

For  use  the  medium  is  distributed  in  Petri  capsules  in  a  rather  thicker 
layer  than  is  customary  in  an  ordinary  plate.  This  sheet  of  medium 
must  be  transparent,  but  must  not  be  less  than  2  mm.  in  thickness — in 
fact,  ought  to  be  about  4  mm.  After  being  poured,  the  capsules  are  left 
with  the  covers  off  for  an  hour  or  so,  to  allow  the  superficial  layers  of 
the  medium  to  become  set  hard.  The  effect  of  this  is  that  during  in- 
cubation no  water  of  condensation  forms  on  the  lid  of  the  capsule,  and 
thus  the  danger  of  this  fluid  dropping  on  to  the  developing  colonies  is 
avoided.  The  antiseptic  nature  of  the  crystal-violet  is  sufficient  to 
prevent  the  growth  of  any  aerial  organisms  falling  on  the  agar  during 
its  exposure  to  the  air.  The  plates  are  usually  inoculated  by  means  of 
a  glass  spatula  made  by  bending  3  inches  of  a  piece  of  glass  rod  at 
right  angles  to  the  rest  of  the  rod.  This  part  is  dipped  in  the  infective 
material,  and  smeared  in  all  directions  over  the  surfaces  of  three  or  four 
plates  successively  without  any  intervening  sterilisation.  The  plates  are 
again  exposed  to  the  air  after  inoculation  for  half  an  hour,  and  then 
incubated  for  twenty-four  hours.  At  the  end  of  such  a  period  b.  coli 
colonies  are  2  to  6  mm.  in  diameter,  stained  distinctly  red,  and  are  non- 
transparent.  Colonies  of  the  b.  typhosus  are  seldom  larger  than  2  mm., 
they  are  blue  or  bluish-violet  in  colour,  are  glassy  and  dew-like  in 
character,  and  have  a  single  contour.  Sometimes  in  the  plates  b.  subtilis 
and  its  congeners  appear,  and  colonies  of  these  organisms  have  a  blue 
colour.  Their  growth  is,  however,  more  exuberant  than  that  of  the 
typhoid  bacillus, — being  often  heaped  up  in  the  centre, — and  the 
contour  of  the  colony  is  often  double. 


J  The  litmus  solution  is  made  as  follows  :  Solid  commercial  litmus  is 
digested  with  pure  spirit  at  30°  C.  till  on  adding  fresh  alcohol  the  latter 
becomes  only  of  a  light  violet  colour.  A  saturated  solution  of  the  residue  is 
then  made  in  distilled  water  aud  filtered.  When  this  is  diluted  with  a  little 
distilled  water  it  is  of  a  violet  colour,  which  further  dilution  turns  to  a  pure 
blue.  To  such  a  blue  solution  very  weak  sulphuric  acid  (made  by  adding 
two  drops  of  dilute  sulphuric  acid  to  200  c.c.  water)  is  added  till  the  blue 
colour  is  turned  to  a  wine-red.  Then  the  saturated  solution  of  the  dye  is 
added  till  the  blue  colour  returns. 


MEDIA  FOR  SEPARATING  BACTERIAL  GROUPS     49 

Conradi's  Picric  Acid  Brilliant  Green  Method.— Applying  his  principle 
of  seeking  for  anilin  bodies  which  while  inhibiting  the  action  of  ordinary 
intestinal  bacteria  rather  favour  the  growth  of  b.  typhosus  and  b.  para- 
typhosus,  Conrad i  in  1908  used  for  this  purpose  crystalline  brilliant 
green  (Hoechst,  extra  pure),  acting  along  with  picric  acid  (Griibler).  The 
medium  is  made  as  follows  :  900  c.c.  water,  20  grins.  Liebig's  meat  extract 
and  100  c.c.  of  a  10  per  cent,  watery  solution  of  Witte's  peptone  are 
mixed  and  filtered  ;  30  grms.  agar  in  threads  are  dissolved  in  the  Huid,  and 
the  whole  filtered.  The  reaction  is  then  adjusted  with  normal  sodium 
hydrate  or  normal  phosphoric  acid  to  an  acid  content  of  3  per  cent, 
(phenol-phthaleine  being  the  indicator),  i.e.  the  finished  medium  is 
such  that  to  make  it  neutral  would  require  the  addition  to  each  100 
c.c.  of  3  c.c.  of  normal  sodium  hydrate.  The  acid  medium  is  then 
sterilised,  and  kept  in  bulk  in  this  form.  For  use  the  remaining 
substances  are  added  in  the  proportions  of  10  c.c.  of  1-1000  watery 
solution  of  the  brilliant  green  and  10  c.c.  of  1  per  cent,  watery  picric 
acid  to  1^  litres  of  the  peptone-agar,  and  the  finished  medium  is  poured 
in  large  Petris  and  allowed  to  stand  at  37°  C.  till  the  surface  is  firm. 
The  capsules  are  inoculated  in  the  wajr  already  described.  Typhoid 
colonies  appear  sharp-edged,  round,  flat-surfaced  but  slightly  thicker 
in  the  middle,  transparent,  and  of  light  green  colour.  Colonies  of  the 
paratyphoid  bacillus  are  similar,  but  tend  at  the  same  age  to  be  slightly 
larger  and  have  a  somewhat  yellowish  green  tint. 

Fawcus's  Picric  Acid  and    Brilliant    Green    Medium.— This    is    a 

modification  of  Conradi's  medium  which  has  been  used  with  great 
success  at  the  Royal  Army  Medical  College  in  the  investigation  of 
typhoid  carriers.  It  is  made  as  follows :  To  900  c.c.  tap  water  add 
5  grms.  sodium  taurocholate  (which  is  commercially  prepared  from  ox 
bile),  30  grms.  powdered  agar,  30  grms.  Witte's  peptone,  5  grms.  sodium 
chloride  ;  steam  for  three  hours,  clear  with  white  of  egg,  filter  through 
cotton  wool,  and  bring  to  a  reaction  of  + 15  with  normal  lactic  acid  or 
caustic  soda,  and  sterilise.  Dissolve  10  grms.  lactose  in  100  c.c.  sterile 
distilled  water,  and  add  to  melted  agar.  Mix  and  filter  through  Chardin 
paper,  sterilise  carefully,  and  store  in  100  c.c.  flasks.  For  use,  add  to 
each  100  c.c.  flask  2  c.c.  of  a  1-1000  watery  solution  of  brilliant  green 
and  '2  c.c.  of  a  1  per  cent,  watery  solution  of  picric  acid.  Pour  into 
large  Petri  dishes,  and  leave  these  to  stand  inverted  at  37°  C.  till  the 
surface  hardens.  Inoculate  as  usual.  Colonies  of  b.  typhosus  of  twenty- 
four  hours'  growth  are  of  about  1  mm.  in  diameter,  transparent  and  re- 
fracting -  those  of  b.  coli,  on  the  other  hand,  have  a  deep  green  centre, 
though  later  typhoid  colonies  may  also  present  a  pale  green  centre. 

In  the  case  of  several  of  the  special  media  used  for  the  isolation  of 
typhoid  bacilli  under  circumstances  where  other  bacteria  are  present,  a 
difficulty  arises  from  the  fact  that  the  agglutinability  of  the  strains 
isolated  appears  to  be  affected  by  substances  present  in  the  media. 
The  application  of  this  important  confirmatory  diagnostic  method  is  thus 
interfered  with.  This  is  said  not  to  occur  with  the  Conradi  brilliant 
given  method,  and  we  have  found  with  this  medium  that,  if  typhoid 
colonies  do  not  at  once  clump  with  typhoid  serum,  daily  sub-culture  on 
ordinary  agar  yields  in  a  few  days  a  culture  to  which  the  agglutination 
test  can  be  applied. 

Endo's  Medium. — This  is  another  of  the  modern  media  introduced  for 
facilitating  the  separation  of  the  b.  typhosus  from  stools,  etc.  It  is 
made  as  follows:  A  litre  of  3  per  cent,  agar  is  prepared  with  the  usual 


50     METHODS  OF  CULTIVATION  OF  BACTERIA 

constituents,  and  is  boiled,  filtered,  and  rendered  neutral.  It  is  then 
made  alkaline  by  the  addition  of  10  c.c.  of  a  10  per  cent,  solution  of 
sodium  hydrate,  and  there  are  added  10  grammes  of  chemically  pure  milk 
sugar  (free  from  cane  sugar)  and  5  c.c.  of  a  filtered  saturated  alcoholic 
solution  of  basic  fuchsin.  After  thorough  mixing  there  is  added  25  c.c. 
of  a  freshly  prepared  10  per  cent,  solution  of  sodium  sulphite,  the  effect 
of  this  step  being  to  remove  the  colour  of  the  fuchsin  so  that  the  finished 
medium  when  cool  is  quite  colourless.  Of  the  medium  15  c.c.  are  placed 
in  each  of  a  number  of  tubes,  these  are  steamed  for  fifteen  minutes  and 
must  then  be  kept  in  the  dark.  For  use  the  contents  of  a  tube  are 
poured  into  a  sterile  Petri  capsule,  allowed  to  set  in  a  still,  dustless 
atmosphere,  and  are  then  inoculated  as  in  the  other  methods  described. 
After  twenty-four  hours'  growth  colonies  of  b.  coli  appear  red,  while  those 
of  b.  typhosus  are  colourless.  Endo  also  claims  for  his  medium  that 
typhoid  bacilli  isolated  by  its  means  are  agglutinable  by  a  typhoid 
serum.  The  rationale  of  the  colour  reaction  appears  to  be  that  fuchsin, 
which  is  rosanilin  hydrochlorate  (C20H19"N3HC1),  is  reduced  to  rosanilin 
(a  colourless  substance)  by  the  sodium  sulphite.  This  colourless  base 
produces  a  red  colour  with  acids,  such  as  the  lactic  acid  formed  by  the 
b.  coli  in  its  fermentation  of  lactose. 

MacConkey's  Bile-Salt  Media.— These  media  were  introduced  for  the 
purpose  of  differentiating  the  intestinal  bacteria,  and  have  been  exten- 
sively used  for  the  study  of  the  b.  coli,  b.  typhosus,  b.  dysenteric,  etc. 
The  characteristic  ingredients  are  bile  salts  and  various  sugars.  The 
stock  solution  is  the  following:  Commercial  sodium  taurocholate,  '5 
gramme  ;  Witte's  peptone,  2'0  grammes;  tap  water,  100  c.c.  (if  distilled 
water  be  used,  '03  per  cent,  of  calcium  chloride  should  be  added).  The 
solution  is  steamed  for  two  hours,  filtered  when  hot,  allowed  to  stand  for 
twenty-four  hours  or  till  sedimentation  has  occurred, and  filtered  again.  For 
a  liquid  medium  there  is  added  to  this  "25  per  cent,  of  a  freshly  prepared 
1  per  cent,  solution  of  neutral  red J  and  the  sugar, — when  glucose,  dulcite, 
or  adonite  is  used,  '5  per  cent,  is  added,  in  the  case  of  other  sugars 
1  per  cent.  The  fluid  is  distributed  in  Durham's  fermentation  tubes  and 
sterilised  in  the  steamer  for  ten  minutes  on  two  successive  days,  care 
being  taken  not  to  overheat  the  medium. 

For  bile-salt  agar  1*5  to  2  per  cent,  agar  is  dissolved  in  the  stock 
solution  in  the  autoclave,  if  necessary  cleared  with  white  of  egg  and 
filtered.  Neutral  red  and  a  sugar  are  added,  as  in  the  case  of  the  liquid 
medium.  As  with  Drigalski's  medium,  it  is  well  to  sterilise  it  in  flasks 
containing  80  c.c.,  this  being  an  amount  sufficient  for  three  Petri 
capsules.  When  this  medium  is  used  for  examining  urine  or  faeces, 
plates  are  inoculated  as  with  Drigalski's  medium  (supra}  ;  for  its  use  in 
water  examinations,  see  p.  157. 

With  reference  to  their  behaviour  in  MacConkey's  fluid  medium  with 
glucose,  organisms  are  divided  into  (1)  those  which  produce  both  acid  and 
gas  ;  (2)  those  producing  acid  only  ;  (3)  those  growing  but  not  producing 
either  acid  or  gas  ;  (4)  those  incapable  of  growing.  B.  coli  belongs  to  the 
first  group  and  b.  typhosus  to  the  second,  and  to  these  groups  also  belong 
most  ordinary  organisms  growing  in  faeces,  practically  none  of  which  are 
found  in  the  third  and  fourth  classes.  Further,  a  number  of  ordinary  non- 


1  The  use  of  neutral  red  in  a  bile-salt  medium  was  first  introduced  by 
Griinbaum  and  Hume.  It  gives  a  deep  crimson  with  acids  and  a  yellow-brown 
with  alkalies. 


MEDIA  FOR  SEPARATING  BACTERIAL  GROUPS     51 

pathogenic  organisms  and  also  some  that  are  pathogenic  have  their  free 
growth  inhibited  in  bile-salt  media.  Thus,  if  any  growth  takes  place 
on  this  medium  when  inoculated  with,  say,  water,  the  probability  is  that 
the  bacteria  have  been  derived  from  faices,  but  of  course  further  procedures 
for  their  identification  must  be  undertaken. 

When  growth  of  a  bacterium  producing  acid  and  gas  occurs  in  neutral- 
red  fluid  media  the  latter  turns  a  rose  colour,  and  gas  appears  in  the 
Durham's  tube.  Sometimes  a  fluorescent  appearance  is  also  observed, 
the  significance  of  which  will  be  discussed  in  the  chapter  on  b.  coli. 
With  the  neutral-red  solid  media  the  colonies  of  any  organism  giving 
rise  to  acid  will  be  of  a  rose-red  colour. 

Petruschky's  Litmus  Whey. — The  preparation  of  this  medium,  which 
is  somewhat  difficult,  is  as  follows :  Fresh  milk  is  slightly  warmed, 
and  sufficient  very  dilute  hydrochloric  acid  is  added  to  cause  precipita- 
tion of  the  casein,  which  is  now  filtered  off.  Dilute  sodium  carbonate 
solution  is  added  up  to,  but  not  beyond,  the  point  of  neutralisation,  and 
the  fluid  steamed  for  one  to  two  hours,  by  which  procedure  any  casein 
which  has  been  converted  into  acid  albumin  by  the  hydrochloric  acid 
is  precipitated.  This  is  filtered  off,  and  a  clear,  colourless,  perfectly 
neutral  fluid  should  result.  Its  chief  constituent,  of  course,  will  be 
lactose.  To  this  sufficient  Kubel-Tiemann  solution  of  litmus  is  added, 
the  medium  is  put  into  tubes  and  then  sterilised.  (This  is  the  original 
method,  but  it  is  better,  after  the  casein  has  been  precipitated,  to  make 
the  medium  slightly  alkaline  with  the  sodium  carbonate  and  bring  to 
the  boiling-point;  then  filter,  neutralise,  add  the  litmus,  and  sterilise.) 
After  growth  has  taken  place,  the  amount  of  acid  formed  can  be  estimated 
liy  dropping  in  standardised  soda  solution  till  the  tint  of  an  uninoculated 
tube  is  readied. 

Eisner's  Medium. — This  is  another  of  the  media  introduced  in  the 
study  of  the  comparative  reactions  of  the  typhoid  bacillus  and  the  b.  coli. 
The  preparation  is  as  follows  :  500  grammes  potato  arc  grated  up  ill  a 
litre  of  water,  allowed  to  stand  over  night,  then  strained,  and  added  to 
an  equal  quantity  of  ordinary  15  per  cent,  peptone  gelatin  which  has  not 
been  neutralised.  Normal  sodium  hydrate  solution  is  added  till  the 
reaction  is  feebly  acid  to  litmus,  the  whole  boiled  together,  filtered,  and 
sterilised.  Just  before  use  potassium  iodide  is  added  so  as  to  constitute 
1  per  cent,  of  the  medium.  Moore  has  used  a  similar  agar  preparation. 
Here  500  grammes  potato  are  scraped  up  in  one  litre  of  water,  allowed  to 
stand  for  three  hours,  strained,  and  put  aside  over  night.  The  clear 
fluid  is  poured  oil',  made  up  to  one  litre,  rendered  slightly  alkaline,  20 
grammes  agar  are  added,  and  the  whole  is  treated  as  in  making  ordinary 
agar.  The  medium  is  distributed  in  test-tubes — 10  c.c.  to  each — and 
immediately  before  use,  to  each  is  added  *5  c.c.  of  a  solution  of  10 
gramnif>  j.ntassium  iodide  to  50  c.c.  water. 

Any  one  of  these  media  in  the  hands  of  a  worker  accustomed 
to  its  use  will  yield  good  results.  MacConkey's  medium  is  that 
most  used  by  British  workers,  and  it  has  the  merit  of  being 
i-;i>ily  prepared.  As  the  result  of  a  considerable  experience 
we  have  found  it  most  useful  and  reliable.  Next  to  it  \\« 
would  place  r'a\vi-us's  modification  of  Conradi's  brilliant  green 
method. 


52      METHODS  OF  CULTIVATION  OF  BACTERIA 


Media  for  growing  Trichophyta,  Moulds,  etc. 

1.  Beer  Wort  Agar.—  Take  beer  wort  as  obtainable  from  the  brewery, 
and  dilute  it  till  it  has  an  s.g.  of  1100.     Add  1'5  per  cent,  of  powdered 
agar,  and  heat  in  the  Koch  till  it  is  dissolved  (usually  about  two  hours 
are   necessary).     Filter  rapidly  and  fill  into  tubes.     Sterilise  in  the  Koch 
for  twenty  minutes  on  three  successive  days.     If  the  medium  is  heated  too 
long  it  loses  the  capacity  of  solidifying. 

2.  Sabouraud's     Media. — Sabouraud     recommends     the      following 
media  : — 

(1)  Distilled  water 1000  c.c. 

Maltose  ("brute  de  Chanut")       .         .         .  40  grms. 

Peptone  ("granulee  de  Chassaing")     .         .  10     ,, 

Agar    .         .                  18     ,, 

(2)  Distilled  water 1000  c.c. 

Glucose  ("massee  de  Chanut")     .         .         .         40  grms. 
Peptone  ("granule  de  Chassaing")     .         .         10     ,, 
Agar    .         .         .         .         .         .  '      .        .         18     ,, 

In  each  case  the  ingredients  are  mixed  and  dissolved  by  gradually 
raising  to  120°  C.  in  an  autoclave.  The  medium  is  then  rapidly  filtered 
through  papier  chardin  (Cogit,  36  Boulevard  Saint  Michel,  Paris)  ; 
when  the  filtrate  begins  only  to  pass  in  drops  the  fluid  is  transferred  to 
another  filter,  and  this  is  repeated  as  often  as  is  necessary.  The  medium 
is  distributed  in  wide  test-tubes  or  Erlerimeyer's  flasks,  plugged  with  non- 
absorbent  cotton  wool,  and  sterilised  by  slowly  raising  the  temperature 
to  120°  C. 

To  use  these  for  isolating,  say,  the  Tinea  tonsurans,  pick  out  an 
infected  hair,  wash  in  absolute  alcohol  for  a  few  seconds,  then  wash  in 
changes  of  sterile  waterj;  cut  into  small  pieces  and  place  these  on  the 
surface  of  the  medium  ;  incubate  at  24°  C.  Usually,  however,  it  is  un- 
necessary to  disinfect  .hair  or  skin  scales  from  which  dermophyta  are  to 
be  isolated. 

THE  USE  OF  THE  ORDINARY  CULTURE  MEDIA. 

The  culture  of  bacteria  is  usually  carried  on  in  test-tubes 
conveniently  6  x  f  in.  These  ought  to  be  very  thoroughly 
washed  and  dripped,  and  their  mouths  plugged  with  plain 
cotton  wool.  They  are  then  sterilised  for  one  hour  at  170°  C. 
If  the  tubes  be  new,  the  glass,  being  usually  packed  in  straw, 
may  be  contaminated  with  the  extremely  resisting  spores  of 
the  b.  subtilis.  Cotton-wool  plugs  are  universally  used  for 
protecting  the  sterile  contents  of  flasks  and  tubes  from  con- 
tamination with  the  bacteria  of  the  air.  A  medium  thus 
protected  will  remain  sterile  for  years.  Whenever  a  protecting 
plug  is  removed  for  even  a  short  time,  the  sterility  of  the 
contents  may  be  endangered.  It  is  well  to  place  the  bouillon, 
gelatin,  and  agar  media  in  the  test-tubes  directly  after  filtration. 
The  media  can  then  be  sterilised  in  the  test-tubes. 


THE  USE  OF  THE  ORDINARY  CULTURE  MEDIA     53 

In  filling  tubes,  care  must  be  taken  to  run  the  liquid  down 
the  centre,  so  that  none  of  it  drops  on  the  inside  of  the  upper 
part  of  the  tube  with  which  the  cotton-wool  plug  will  be  in 
contact,  otherwise  the.  latter  will  subsequently  stick  to 
the  glass  and  its  removal  will  be  difficult.  In  the  case  of 
liquid  media,  test-tubes  are  filled  about  one-third  full.  With 
the  solid  media  the  amount  varies.  In  the  case  of  gelatin 


A  e 

FIG.  13.— Tubes  of  media. 


The  apparatus  explains  itself. 
The  indiarubber  stopper  with 
its  tubes  ought  to  be  steril- 
ised before  use. 

media,  tubes  filled  one-third  full  and  allowed  to  solidify 
while  standing  upright,  are  those  commonly  used.  With 
organisms  needing  an  abundant  supply  of  oxygen  the  best 
growth  takes  place  on  the  surface  of  the  medium,  and  for 
practical  purposes  the  surface  ought  thus  to  be  as  large  as 
possible.  To  this  <end  "  sloped "  agar  and  gelatin  tubes  are 
used.  To  prepare  these,  tubes  are  filled  only  about  one-sixth 
full,  and  after  sterilisation  are  allowed  to  solidify,  lying  on  their 
sides  with  their  necks  supported  so  that  the  contents  extend 


54      METHODS  OF  CULTIVATION  OF  BACTERIA 

3  to  4  inches  up,  giving  an  oblique  surface  after  solidification. 
Thus  agar  is  commonly  used  in  such  tubes  (less  frequently 
gelatin  is  also  "  sloped  "),  and  this  is  the  position  in  which  blood 
serum  is  inspissated.  Tubes,  especially  those  of  the  less  commonly 
used  media,  should  be  placed  in  large  jars  provided  with  stoppers, 
otherwise  the  contents  are  apt  to  evaporate.  A  tube  of  medium 
which  has  been  inoculated  with  a  bacterium,  and  on  which 
growth  has  taken  place,  is  called  a  "  culture."  A  "  pure  culture  " 
is  one  in  which  only  one  organism  is  present.  The  methods  of 
obtaining  pure  cultures  will  presently  be  described.  When  a 
fresh  tube  of  medium  is  inoculated  from  an  already  existing 
culture,  the  resulting  growth  is  said  to  be  a  "  sub-culture  "  of  the 
first.  All  manipulations  involving  the  transference  of  small 
portions  of  growth  either  from  one  medium  to  another,  as  in  the 


FIG.  14. — Platinum  wires  in  glass  handles. 

a.  Straight  needle  for  ordinary  puncture  inoculations,     b.  "Platinum  loop." 
c.  Long  needle  for  inoculating  "  deep  "  tubes. 

inoculation  of  tubes,  or,  as  will  be  seen  later,  to  cover-glasses  for 
microscopic  examination,  are  effected  by  pieces  of  platinum  wire 
(Nos.  24  or  27  Birmingham  wire  gauge — the  former  being  the 
thicker)  fixed  in  glass  rods  8  inches  long.1  Every  worker  should 
have  three  such  wires.  Two  are  2J  inches  long,  one  of  these 
being  straight  (Fig.  14,  a),  and  the  other  having  a  loop  turned 
upon  it  (Fig.  14,  b).  The  latter  is  referred  to  as  the  platinum 
"loop"  or  platinum  "eyelet,"  and  is  used  for  many  purposes. 
"  Taking  a  loopful "  is  a  phrase  constantly  used.  The  third  wire 
(Fig.  14,  c)  ought  to  be  4^  inches  long  and  straight.  It  is  used 
for  making  anaerobic  cultures.  It  is  also  very  useful  to  have 
at  hand  a  platinum-iridium  spud.  This  consists  of  a  piece  of 
platinum-iridium  about  1J  inches  long,  2  mm.  broad,  and  of 
sufficient  thickness  to  give  it  a  firm  consistence ;  its  distal  end  is 

1  Aluminium  rods  are  made  which  are  very  convenient.  The  end  is  split 
with  a  knife,  the  platinum  wire  is  inserted  and  fixed  by  pinching  the 
aluminium  on  it  in  a  vice, 


THE  USE  OF  THE  ORDINARY  CULTURE  MEDIA     55 


expanded  into  a  diamond  shape,  and  its  proximal  is  screwed 
into  an  aluminium  rod.  It  is  very  useful  for  making  scrapings 
from  organs  and  for  disintegrating  felted  bacterial  cultures ;  in 
such  manipulations  the  ordinary  platinum  wire  is  awkward  to 
work  with,  as  it  bends  so  easily.  Cultures  on  a  solid  medium 
are  referred  to  (1)  as  "puncture"  or  "stab"  cultures  (German, 
Stichkultur),  or  (2)  as  "stroke"  or  "  slant "  cultures  (Strichkultur), 
according  as  they  are  made  (1)  on  tubes  solidified  in  the  upright 
position,  or  (2)  on  sloped  tubes. 

To  inoculate,1  say,  one  ordinary  upright  gelatin  tube  from 
another,  the  two  tubes  are  held  in  an  inverted  position  between 
the  forefinger  and  thumb  of  the  left  hand  with  their  mouths 
towards  the  j>erson  holding  them ;  the  plugs  are  twisted  round 
once  or  twice,  to  make  sure  they  are  not  adhering  to  the  glass. 
The  short,  straight  platinum  wire  is  then  heated  to  redness  from 
point  to  insertion,  and  2  to  3  inches  of  the  glass  rod  are  also 
passed  two  or  three  times  through  the  Bunsen  flame.  It  is  held 
between  the  right  fore  and  middle  fingers,  with -the  needle  pro- 
jecting backwards,  i.e.  away  from  the  right  palm.  Remove  plug 
from  culture  tube  with  right  forefinger  and  thumb,  and  continue 
to  hold  it  between  the  same  fingers  by  the  part  which  projected 
beyond  the  mouth  of  the  tube.  Now  touch  the  culture  with  the 
platinum  needle,  and,  withdrawing  it,  replace  plug.  In  the 
same  way  remove  plug 
from  tube  to  be  in- 
oculated, and  plunge 
platinum  wire  down 
the  centre  of  the 
gelatin  to  within  half 
an  inch  of  the  bottom. 
It  must  on  no  account 
touch  the  glass  above 
the  medium.  The  wire 
is  then  immediately 
sterilised.  A  variation 
in  detail  of  this  method 
is  to  hold  the  plug  of 
the  tube  next  the 
thumb  between  the  fore 
and  middle  fingers,  and 

the  plug  of  the  other  between  the  middle  and  ring  fingers,  then 
to  make  the  inoculation  (Fig.  15).  If  a  tube  contain  a  liquid 
medium,  it  must  be  held  in  a  sloping  position  between  the  same 
fingers,  as  above.  For  a  stroke  culture  the  platinum  loop  is 


FIG.  15. — Another  method  of  inoculating 
solid  tubes. 


56      METHODS  OF  CULTIVATION  OF  BACTERIA 

used,  and  a  little  of  the  culture  is  smeared  in  a  line  along  the 
surface  of  the  medium  from  below  upwards.  In  inoculating 
tubes,  it  is  always  well,  on  removing  the  plugs,  to  make  sure 

that  no  strands  of  cotton 
fibre  are  adhering  to  the 
inside  of  the  necks.  As 
these  might  be  touched  with 
the  charged  needle  and  the 
plug  thus  be  contaminated, 
they  must  be  removed  by 
heating  the  inoculating 
needle  red-hot  and  scorch- 
FIG.  16.— Rack  for  platinum  needles.  ing  them  off  with  it.  When 

the  platinum  wires  are  not 

in  use  they  may  be  laid  in  a  rack  made  by  bending  up  the  ends 
of  a  piece  of  tin,  as  in  Fig.  16.  To  prevent  contamination  of 
cultures  by  bacteria  falling  on  the  plugs  while  these  are  exposed 
to  the  air  during  inoculation  manipulations,  some  bacteriologists 
singe  the  plugs  in  the  flame  before  replacing.  This  is,  however, 
in  most  cases  a  needless  precaution.  If  the  top  of  a  plug  be 
dusty  it  is  best  to  singe  it  before  extraction. 

THE  METHODS  OF  THE  SEPARATION  OF  AEROBIC  ORGANISMS. 
PLATE  CULTURES. 

The  general  principle  underlying  the  methods  of  separation 
is  the  distribution  of  the  bacteria  in  one  of  the  solid  media 
liquefied  by  heat  and  the  dilution  of  the  mixture  so  that  the 
growths  produced  by  the  individual  bacteria — called  colonies — 
shall  be  suitably  apart.  In  order  to  render  the  colonies  easily 
accessible,  the  medium  is  made  to  solidify  in  as  thin  a  layer  as 
possible,  by  being  poured  out  on  glass  plates — hence  the  term 
"plate  cultures." 

As  the  optimum  temperature  varies  with  different  bacteria, 
it  is  necessary  to  use  both  gelatin  and  agar  media.  Many 
pathogenic  organisms,  e.g.  pneumococcus,  b.  diphtheriae,  etc., 
grow  too  slowly  on  gelatin  to  allow  its  ready  use.  On  the  other 
hand,  many  organisms,  e.g.  some  occurring  in  water,  do  not 
develop  on  agar  incubated  at  37°  C. 

Separation  by  Gelatin  Media. — As  the  naked-eye  and  micro- 
scopic appearances  of  colonies  are  often  very  characteristic, 
plate  cultures,  besides  use  in  separation,  are  often  taken 
advantage  of  in  the  description  of  individual  organisms.  The 
plate-culture  method  can  also  be  used  to  test  whether  a  tube 


SEPARATION  BY  GELATIN  MEDIA  57 

culture  is  or  is  not  pure.  The  suspected  culture  is  plated  (three 
plates  being  prepared,  as  will  be  described).  If  all  the  colonies 
are  the  same,  then  the  culture  may  be  held  to  be  pure. 

Either  simple  plates  of  glass  4  inches  by  3  inches  are  used, 
or,    what    are    more     convenient, 
circular    glass    cells    with    similar 
overlapping    covers.       The    latter 
are    known   as   Petri's    dishes    or 
capsules    (Fig.     17).       They    are 
usually  3  inches  in  diameter  and 
half  an  inch  deep.     The  advant- 
age of  these  is  that  they  do  not         FIG.  17. -^tri's  capsule, 
require    to     be     kept    level    by    a      (Cover  shown  partially  raised.) 
.special  apparatus  while  the  medium 

is  solidifying,  and  can  be  readily  handled  afterwards  without 
admitting  impurities.  Whether  plates  or  capsules  are  used, 
they  are  washed,  dried  with  a  clean  cloth,  and  sterilised  for  one 
hour  in  dry  air  at  170°  C.,  the  plates  being  packed  in  sheet-iron 
boxes  made  for  the  purpose  (see  Fig.  18). 

1.  Glass  Capsules. — While  in  certain  circumstances,  as  when 
the  number  of  colonies  has  to  be  counted,  it  is  best  to  use  plates 
of  glass,  Petri's  capsules  are  to  be  preferred  in-  the  usual  labora- 
tory routine  for  the  above  reasons. 

The  contents  of  three  gelatin  tubes,  marked  a,  b,  c,1  are 
liquefied  by  placing  in  a  beaker  of  water  at  any  temperature 
between  25°  C.  and  38°  C.  Inoculate  a  with  the  bacterial 
mixture.  The  amount  of  the  latter  to  be  taken  varies,  and  can 
only  be  regulated  by  experience.  If  the  microscope  shows 
enormous  numbers  of  different  kinds  of  bacteria  present,  just  as 
much  as  adheres  to  the  point  of  a  straight  platinum  needle  is 
suilicient.  If  the  number  of  bacilli  is  small,  one  to  three  loops 
of  the  mixture  may  be  transferred  to  the  medium.  Shake  a 
wrll,  but  not  so  as  to  cause  many  fine  air-bubbles  to  form. 
Transfer  two  loops  of  gelatin  from  a  to  b.  Shake  b  and  transfer 
five  loops  to  c.  The  plugs  of  the  tubes  are  in  each  case  replaced 
and  the  tubes  returned  to  the  beaker.  The  contents  of  the 
three  tubes  are  then  poured  out  into  three  capsules.  In  doing 
so  the  plug  of  each  tube  is  removed  and  the  mouth  of  the  tube 
passed  two  or  three  times  through  the  Bunsen  flame,  the  tube 
being  meantime  rotated  round  a  longitudinal  axis.  Any  organ- 
isms on  its  rim  are  thus  killed.  The  capsules  are  labelled  and 
set  aside  till  growth  takes  place. 

1  For  marking  glass  vessels  it  is  convenient  to  use  the  red,  blue,  or  yellow 
oil  pencils  made  for  the  purpose  by  Faber. 


58      METHODS  OF  CULTIVATION  OF  BACTERIA 


For  accurate  work  it  will  be  found  convenient  to  carry  out 
the  dilutions  in  definite  proportions.     The  following  is  the  pro- 


FlG.  18. — Koch's  levelling  apparatus  for  use  in  preparing  plates. 
Hands  shown  in  first  position  for  transferring  sterile  plate  from  iron 
box  to  beneath  bell  jar,  where  it  subsequently  has  the  medium  poured 
out  upon  it. 

cedure  which  we  .have  found  very  serviceable  :  In  a  number  of 
small  sterile  test-tubes  '95  c.c.  sterile  water  is  put.     To  the  first 


FIG.  19.- — Koch's  levelling  apparatus.  Hands  shown  in  second 
position  just  as  the  plate  is  lowered  on  to  the  ground  glass  surface. 
By  executing  the  transference  of  the  plate  from  the  box  in  this  way, 
the  surface  which  was  undermost  in  the  latter  is  uppermost  in  the 
leveller,  and  thus  never  meets  a  current  of  air  which  might  con- 
taminate it. 

tube  we  add  '05  c.c.  of  the  bacterial  mixture.     The  contents  of 
the  tube  are  well  shaken  up,  and  the  pipette  is  sterilised  by 


SEPARATION  BY  GELATIN  MEDIA  59 

being  washed  out  with  boiling  water.  It  is  allowed  to  cool,  and 
•05  c.c.  of  fluid  is  transferred  from  the  first  tube  to  the  second. 
By  a  similar  procedure  '05  c.c.  is  transferred  from  the  second  to 
the  third,  and  so  on.  There  is  thus  effected  a  twenty-fold 
dilution  in  each  successive  tube.  After  these  steps  have  been 
carried  out,  a  definite  amount,  say,  '05  c.c.,  is  transferred  from 
each  tube  to  a  tube  of  melted  gelatin, — the  gelatin  being  after- 
wards plated  and  the  colonies  counted  when  growth  occurs. 
The  number  of  tubes  required  will  vary  according  to  the 
number  of  bacteria  in  the  original  mixture,  but  usually  four  or 
five  will  be  sufficient.  It  is  quite  evident  that  this  method  not 
only  enables  us  to  separate  bacteria,  but  if  necessary  gives  us  a 
means  of  estimating  exactly  the  number  in  the  original  mixture. 
The  colonies  appear  as  minute  rounded  points,  whitish  or 
variously  coloured.  Their  characters  can  be  more  minutely 
studied  by  means  of  a  hand-lens  or  by  inverting  the  capsule  on 
the  stage  of  a  microscope  and  examining  with  a  low  power 
through  the  bottom.  From  their  characters,  colour,  shape, 
contour,  appearance  of  surface,  liquefaction  or  non-liquefaction 
of  the  gelatin,  etc.,  the  colonies  can  be  classified  into  groups. 
Further  aid  in  the  grouping  of  the  varieties  is  obtained  by 
making  film  preparations  and  examining  them  microscopically. 
Gelatin  or  agar  tubes  may  then  be  inoculated  from  a  colony  of 
each  variety,  and  the  growths  obtained  are  then  examined  both 
as  to  their  purity  and  as  to  their  special  characters,  with  a  view 
to  their  indentification  (p.  137). 

2.  Glass  Plates  (Koch). — When  plates  of  glass  are  to  be  used,  an 
apparatus  on  which  they  may  be  kept  level  while  the  medium  is  solidi- 
fying is,  as  has  been  said,  necessary.  An  apparatus  devised  by  Koch  is 
used  (Figs.  18,  19).  This  consists  of  a  circular  plate  of  glass  "(with  the 
upper  surface  ground,  the  lower  polished),  on  which  the  plate  used  for 
pouring  out  the  medium  is  placed.  The  latter  is  protected  from  the  air 
daring  solidification  by  a  bell  jar.  The  circular  plate  and  bell  jar  rest 
<>ii  the  tlat  rim  of  a  circular  glass  trough,  which  is  filled  quite  full  with 
a  mixture  of  ice  and  water,  to  facilitate  the  lowering  of  the  temperature 
of  whatever  is  placed  beneath  the  bell  jar.  The  glass  trough  rests  on 
corks  on  the  bottom  of  a  large  circular  trough,  which  catches  any  water 
that  may  be  spilled.  This  trough  in  turn  rests  on  a  wooden  triangle 
with  a  foot  at  each  corner,  the  height  of  which  can  be  adjusted,  and 
which  thus  constitutes  the  levelling  apparatus.  A  spirit  level  is  placed 
where  the  plate  is  to  go,  and  the  level  of  the  ground  glass  plate  thus 
assured.  There  is  also  prepared  a  "damp  chamber,"  in  which  the 
plates  are  to  be  stored  after  being  made.  This  consists  of  a  circular 
glass  trough  with  a  similar  cover.  It  is  sterilised  by  being  washed  out- 
side' and  inside  with  perchloride  of  mercury  1-1000,  and  a  circle  of  filter- 
papcr  nioi.xtened  with  the  same  is  laid  on  its  bottom.  Glass  benches  on 
which  the  plates  may  be  laid  are  similarly  purified. 


60      METHODS  OF  CULTIVATION  OF  BACTERIA 


To  separate  organisms  by  this  method,  three  tubes,  a,  b,  c,  are  inocu- 
lated as  in  using  Petri's  capsules  (p.  57).  The  hands  having  been 
washed  in  perchloride  of  mercury  1-1000  and  dried,  the  plate  box  is 
opened,  and  a  plate  lifted  by  its  opposite  edges  and  transferred  to  the 
levelled  ground  glass  (as  in  Figs.  18,  19).  The  bell  jar  of  the  leveller 
being  now  lifted  a  little,  the  gelatin  in  tube  a  is  poured  out  on  the 
surface  of  the  sterile  plate,  and,  while  still  fluid,  is  spread  by  stroking 
with  the  rim  of  the  tube.  After  the  medium  solidifies,  the  plate  is 
transferred  to  the  moist  chamber  as  rapidly  as  possible,  so  as  to  avoid 
atmospheric  contamination.  In  doing  this,  it  is  advisable  to  have  an 
assistant  to  raise  the  glass  covers.  Tubes  b  and  c  are  similarly  treated, 
and  the  resulting  plates  stacked  in  series  on  the  top  of  a.  The  chamber 
is  labelled  and  set  aside  for  a  few  days  till  the  colonies  appear  on  the 
gelatin  plates.  The  further  procedure  is  of  the  same  nature  as  with 
Petri's  capsules. 

3.  Esmarctis  Roll  Tubes. — Here  the  principle  is  that  of 
dilution  as  before.  In  each  of  three  test-tubes  1J  or  1J  inch  in 
diameter,  gelatin  to  the  depth  of  three-quarters  of  an  inch  is  placed. 
These  are  sterilised.  The  gelatin  is  melted  and 
inoculated  in  series  with  the  bacterial  mixture  as 
in  making  plate  cultures,  but  instead  of  being 
poured  out  it  is  rolled  in  a  nearly  horizontal 
position  under  a  cold  tap  or  on  a  block  of  ice 
till  it  solidifies  as  a  uniformly  thin  layer  on  the 
inside  of  the  tube.  Practically  we  deal  with  a 
cylindrical  sheet  of  gelatin  instead  of  a  flat  one. 
A  convenient  form  of  tube  for  this  method  is 
one  with  a  constriction  a  short  distance  below 
'the  plug  of  cotton  wool  (Fig.  20).  The  great 
disadvantage  of  the  method  is,  that  if  organisms 
liquefying  the  gelatin  be  present,  the  liquefied 
gelatin  contaminates  the  rest  of  the  medium. 

Separation  by  Agar  Media. — 1.  Agar  Plates. 
— The  only  difference  between  the  technique  here 
and  that  with  gelatin  depends  on  the  difference 
in  the  melting-points  of  the  two  media.     Agar, 
we   have    said,   melts   at    98°   C.,   and    becomes 
E    FlG'h?tube     a§am  s°lid  a  little  under  40°  C.     As  it  is  danger- 
for  roll  culture,    ous  to  expose  organisms  to  a  temperature  much 
above  42°  C.,  it  is  necessary  in  preparing  tubes 
of  agar  to  be  used  in  plate  cultures  first  to  melt  the  agar,  by 
boiling  in  a  vessel  of  water  for  a  few  minutes,  and  then   to 
cool  it  to  about  42°  C.  before  inoculating.     The  manipulation 
must   be    rapidly  carried    out,  as   the    margin   of   time,  before 
solidification  occurs,  is  narrow;  otherwise    the    details   are  the 
same  as  for  gelatin.     Esmarch's  tubes  are  not  suitable  for  use 


SEPARATION  BY  AGAR  MEDIA  61 

here,  as  the  agar  does  not  adhere  well  to  the  sides.  If  to  the 
agar  2  per  cent,  of  a  strong  watery  solution  of  pure  gum  arabic 
is  added,  Esmarch's  tubes  may,  however,  be  used. 

2.  Scjmration  I>y  Stroking  Mixture  on  Surface  of  Agar 
Mulia. — The  bacterial  mixture,  instead  of  being  mixed  in  the 
medium,  i*  spread  out  on  its  surface.  The  method  may  be  used 
both  when  the  bacteria  to  be  separated  are  in  a  fluid,  and  when 
contained  in  a  fairly  solid  tissue  or  substance,  such  as  a  piece 
of  diphtheritic  membrane.  In  the  case  of  a  tissue,  for  example, 
a  small  portion  entangled  in  the  loop  of  a  platinum  needle  is 
stroked  in  successive  parallel  longitudinal  strokes  on  sloped 
agar,  the  same  asj)ect  being  brought  in  contact  with  the  agar  in 
all  the  strokes.  Three  strokes  may  be  made  in  each  tube,  and 
three  tubes  are  usually  sufficient.  In  this  process  the  organisms 
on  the  surface  of  the  tissue  are  gradually  rubbed  off,  and  when 
growth  has  taken  place  it  will  be  found  that  in  the  later  strokes 
the  colonies  are  less  numerous  than  in  the  earlier,  and  sufficiently 
far  apart  to  enable  parts  of  them  to  be  picked  off  without  the 
needle  touching  any  but  one  colony.  When,  as  in  the  case  of 
diphtheritic  membrane,  putrefactive  organisms  may  be  present 
on  the  surface  of  the  tissue,  these  can  be  in  great  part  removed 
by  washing  it  well  in  cold  water  previously  sterilised  (vide 
Diphtheria).  In  the  case  of  liquids,  the  loop  is  charged  and 
similarly  stroked.  Tubes  thus  inoculated  must  be  put  in  the 
incubator  in  the  upright  position  and  must  be  handled  carefully, 
so  that  the  condensation  water,  which  is  always  present  in 
incubated  agar  tubes,  may  not  run  over  the  surface.  Agar, 
poured  out  in  a  Petri's  capsule  and  allowed  to  stand  till  firm, 
may  be  used  instead  of  successive  tubes.  Here  a  sufficient 
number  of  strokes  can  be  made  in  one  capsule.  Sloped  blood- 
serum  tubes  may  IK-  used  instead  of  agar.  The  method  is  rapid 
and  easy,  and  gives  good  results. 

Separation  of  Pathogenic  Bacteria  by  Inoculation  of 
Animals. — It  is  found  difficult  and  often  impossible  to  separate 
l»y  ordinary  plate  methods  certain  pathogenic  organisms,  such 
as  b.  tuberculosis,  1>.  mallei,  and  the  pneumococcus,  when  such 
occur  in  conjunction  with  other  bacteria.  These  grow  best  on 
special  media,  and  the  first  two  (especially  the  tubercle  bacillus) 
grow  so  slowly  that  the  other  organisms  present  outgrow  them, 
cover  the  whole  plates,  and  make  separation  impossible.  The 
method  adopted  in  such  cases  is  to  inoculate  an  animal  with 
the  mixture  of  bacilli,  wait  until  the  particular  disease  develops, 
kill  the  animal,  and  with  all  aseptic  precautions  (vide  p.  1  •!">) 
inoculate  tubes  of  suitable  media  from  characteristic  lesion- 


62      METHODS  OF  CULTIVATION  OF  BACTERIA 

situated  away  from  the  seat  of  inoculation,  e.g.  from  spleen 
in  the  case  of  b.  tuberculosis,  spleen  or  liver  in  the  case  of 
b.  mallei,  and  heart  blood  in  the  case  of  pneumococcus. 

Separation  by  killing  Non-spored  Forms  by  Heat. — This  is 
a  method  which  has  a  limited  application.  As  has  been  said, 
the  spores  of  a  bacterium  resist  heat  more  than  the  vegetative 
forms.  When  a  mixture  contains  spores  of  one  bacterium  and 
vegetative  forms  of  this  and  other  bacteria,  then  if  the  mixture 
be  boiled  for  a  few  minutes  all  the  vegetative  forms  will  be 
killed,  while  the  spores  will  remain  alive  and  will  develop 
subsequently.  This  method  can  be  easily  tested  in  the  case  of 
cultivating  b.  subtilis  from  hay  infusion.  A  little  chopped-up 
hay  is  placed  in  a  flask,  of  water,  which  is  boiled  for  about  ten 
minutes.  On  this  being  allowed  to  cool  and  stand,  in  a  day  or 
two  a  scum  forms  on  the  surface,  which  is  found  to  be  a  pure 
culture  of  the  bacillus  subtilis.  The  method  is  also  often  used 
to  aid  in  the  separation  of  b.  tetani,  vide  infra. 

THE  PRINCIPLES  OF  THE  CULTURE  OF  ANAEROBIC 
ORGANISMS. 

All  ordinary  media,  after  preparation,  may  contain  traces  of 
free  oxygen,  and  will  absorb  more  from  the  air  on  standing. 
(1)  For  the  growth  of  anaerobes  this  oxygen  may  be  expelled  by 
the  prolonged  passing  of  an  inert  gas,  such  as  hydrogen,  through 
the  medium  (liquefied  if  necessary).  Further,  the  medium  must 
be  kept  in  an  atmosphere  of  the  same  gas  while  growth  is  going 
on.  (2)  Media  for  anaerobes  may  be  kept  in  contact  with  the 
air,  if  they  contain  a  reducing  agent  which  does  not  interfere 
with  bacterial  growth.  Such  an  agent  takes  up  any  oxygen 
which  may  already  be  in  the  medium,  and  prevents  further 
absorption.  The  reducing  body  used  is  generally  glucose,  though 
formate  of  sodium  may  be  similarly  employed.  The  preparation 
of  such  media  has  already  been  described  (pp.  37,  38).  In  this 
case  the  medium  ought  to  be  of  considerable  thickness. 

The  Supply  of  Hydrogen  for  Anaerobic  Cultures. — The  gas  is  generated 
in  a  large  Kipp's  apparatus  from  pure  sulphuric  acid  and  pure  xi.nc.  It 
is  passed  through  three  wash-bottles,  as  in  Fig.  21.  In  the  first  is 
placed  a  solution  of  lead  acetate  (1  in  10  of  water)  to  remove  any  traces- 
of  sulphuretted  hydrogen.  In  the  second  is  placed  a  1  in  10  solution  of 
silver  nitrate  to  remove  any  arsenietted  hydrogen  which  may  be  present 
if  the  zinc  is  not  quite  pure.  In  the  third  is  a  10  per  cent,  solution 
of  pyrogallic  acid  in  caustic  potash  solution  (1  :  10)  to  remove  any 
traces  of  oxygen.  The  tube  leading  from  the  last  bottle  to  the  vessel 
containing  the  medium  ought  to  be  sterilised  by  passing  through  a 


SEPARATION  OF  ANAEROBIC  ORGANISMS       63 

Bunsen  flame,  and  should  have  a  small  plug  of  cotton  wool  in  it  to  filter 
the  hydrogen  ^enn-l'ree. 

1'i/fOf/allate  of  Potassium  for  Anaerobic  Cultures. — In  arranging  for  the 
absorption  of  oxygen  by  this  substance  the  proportions  used  in  Bulloch's 
-r] .nation  method  may  be  employed.  Here  109  grans,  solid  caustic 
potash  are  dissolved  in  145  c.c.  water,  and  to  this  2-4  grms.  pyrogallol 
art-  added. 

Separation  of  Anaerobic  Organisms. — (a)  By  Roll-tubes. — 
A  1  j  inch  test-tube  has  as  much  gelatin  put  into  it  as  would  be 
used  in  the  Esmarch  roll-tube  method.  It  is  corked  with  an 
indiarubber  stopper  having  two  tubes  passing  through  it,  as  in 
Fig.  "22.  The  ends  of  the  tubes  are  partly  drawn  out  as  shown, 


Fit;.  2L— Apparatus  for  supplying  hydnuren  for  anaerobic  cultures. 

(t.    Ki)>i>'>   apparatus   for  manufacture   of   Imlro.uvn.     l>.    Wash-bottle  con- 

taininu-  1  -It)  solution  ot  lead  :n-etate.     <•.    Wa^h-bolt  I< ulainiii^  1-10  solution 

of  silver  nitrate.     </.  Wash-bottle  containing:  1-10  solution  of  pyro^allic  acid. 
(l>,  c,  and  </  are  intentionally  drawn  to  a  larger  scale  than  n  to  show  details.) 


and  covered  witli  plugs  of  cotton  wool.  Three  such  test-tubes 
HIV  prepared,  and  they  are  sterilised  in  the  steam  steriliser  (p.  28). 
At't'-r  >terilisation  the  gelatin  is  melted  and  one  tube  inoculated 
with  tin-  mixture  containing  the  anaerobes;  the  second  is  inocu- 
lated from  the  first,  and  the  third  from  the  second,  as  in  making 
ordinary  LTriatin  plates.  After  inoculation  the  gelatin  is  kept 
liquid  by  the  lower  ends  of  the  tubes  being  placed  in  water  at 
about  •''»<.)  ('.,  and  hydrogen  is  passed  in  through  tube  ./•  f'-r 
t  unity  minutes.  Tin-  i:as-supply  tubes  are  then  completely 
Sealed  ot!' at  X  and  /,  and  rarli  test-tube  is  rolled  as  in  Ksmareh's 
method  till  the  gelatin  solidities  as  a  thin  layer  on  the  internal 
surface.  A  little  hard  parallin  may  be  run  between  tin-  rim  of  the 


64      METHODS  OF  CULTIVATION  OF  BACTERIA 


containing  anaerobes. 


test-tube  and  the  stopper  and  round  the  .perforations  for  the  gas- 

supply  tubes,  to  ensure  that   the   apparatus    is    airtight.     The 

gelatin  is  thus  in  an  atmosphere  of  hydrogen  in  which  the 
colonies  may  develop.  The  latter  may 
be  examined  and  isolated  in  a  way 
which  will  be  presently  described.  The 
method  is  admirably  suited  for  all 
anaerobes  which  grow  at  the  ordinary 
temperature. 

(b)  Bulloch's  Apparatus  for  An- 
aerobic Culture.  —  This  can  be  recom- 
mended for  plating  out  mixtures 
containing  anaerobes,  and  for  obtaining 
growths  (especially  surface  growths)  of 
the  latter.  It  consists  (Fig.  23)  of  a 
glass  plate  as  base  on  which  a  bell 
jar  can  be  firmly  luted  down  with 
unguentum  resina,  In  the  upper  part 
of  the  bell  jar  are  two  apertures  fur- 
nished with  ground  stoppers,  and 

through  each  of  the  latter  passes  a  glass   tube   on  which  is  a 

stop-cock.     One  tube,  bent  slightly  just  after  passing  through 

the    stopper,   extends   nearly  to    the    bottom   of   the    chamber; 

the  other  terminates  immediately 

below  the  stopper.     In  using  the 

apparatus    there    is    set    on    the 

base  -plate    a    shallow    dish,     of 

slightly  less    diameter  than    that 

of  the  bell  jar,  and  having  a  little 

heap  of  from  2  to  4  grammes  of 

dry  pyrogallic   acid   placed    in  it 

towards  one  side.     Culture  plates 

made   in  the  usual  way  can    be 

stacked  on  a  frame  of  glass  rod's 

resting  on  the  edges  of  the  dish, 

or   a    beaker    containing    culture 

tubes   can  be  placed  in  it.     The 

bell  jar  is  then  placed  in  position 

so  that  the  longer  glass  tube  is 

situated    over    that    part    of    the 

bottom    of    the   shallow    dish   far- 

thest   away   from    the    pyrogallic 

acid,  and  the  bottom  and  stoppers  are  luted.     The  air  in  the 

bell  jar  is  now  expelled  by  passing  a  current  of  hydrogen  through 


.  23.-BullocVs  apparatus  for 
anaerobic  plate  cultures. 


CULTURES  OF  ANAEROBES  65 

the  short  glass  tube,  and  both  stoppers  are  closed.  A  partial 
vacuum  is  then  effected  in  the  jar  by  connecting  up  the  short 
tube  with  an  air-pump,  opening  the  tap,  and  giving  a  few  strokes 
of  the  latter.  A  solution  of  109  grms.  solid  caustic  potash  dis- 
solved in  145  c.c.  water  is  made,  and  into  the  vessel  containing 
it  a  rubber  tube  connected  with  the  long  glass  tube  is  made  to 
dip,  and  the  stopper  of  the  latter  being  opened,  the  fluid  is  forced 
into  the  chamber  and  spreads  over  the  bottom  of  the  shallow 
dish ;  potassium  pyrogallate  is  thus  formed,  which  absorbs  any 
free  oxygen  still  present.  Before  the  whole  of  the  fluid  is  forced 
in  the  rubber  tube  is  placed  in  a  little  boiled  water,  and  this, 
passing  through  the  glass  tubes,  washes  out  the  potash  and 
prevents  erosion  of  the  glass.  The  whole  apparatus  may  be 
placed  in  the  incubator  till  growth  occurs. 

It  is  often  advisable  in  dealing  with  material  suspected  to 
contain  anaerobes  to  inoculate  an  ordinary  deep  glucose  agar 
tube  with  it,  and,  incubating  for  24  or  48  hours,  to  then  apply  an 
anaerobic  separation  method  to  the  resultant  growth.  Sometimes 
the  high  powers  of  resistance  of  spores  to  heat  may  be  taken 
advantage  of  in  aiding  the  separation  (vide  Tetanus). 

Cultures  of  Anaerobes. — When  by  one  or  other  of  the  above 
methods  separate  colonies  have  been  obtained,  growth  may  be 
maintained  on  media  in  contact  with  ordinary  air.  The  media 
generally  used  are  those  which  contain  reducing  agents,  and  the 
test-tubes  containing  the  medium  must  be  filled  to  a  depth  of 
4  inches.  They  are  sterilised  as  usual,  and  are  called  "  deep  " 
tubes.  The  long  straight  platinum  wire  is  used  for  inoculating, 
and  it  is  plunged  well  down  into  the  "  deep  "  tube.  A  little  air 
gets  into  the  upper  part  of  the  needle  track,  and  no  growth  takes 
place  there,  but  in  the  lower  part  of  the  needle  track  growth 
occurs.  From  such  "  deep  "  cultures  growths  may  be  maintained 
indefinitely  by  successive  sub-cultures  in  similar  tubes.  Even 
ordinary  gelatin  and  agar  can  be  used  in  the  same  way  if  the 
medium  is  heated  to  boiling-point  before  use  to  expel  any 
absorbed  oxygen. 

Carroll's  Method  for  Anaerobic  Cultures. — This  may  be  used 
with  culture  tubes  containing  any  of  the  media  suitable  for 
anaerobes,  with  Esmarch's  roll-tubes,  or  with  fermentation  tubes. 
There  is  required  a  dry  tube  of  the  same  diameter  as  the  culture 
tube,  a  short  U-shaped  glass  tube,  and  two  pieces  of  rubber  tub- 
ing all  of  like  diameter.  The  culture  tube  having  been  inoculated, 
the  plug  is  pushed  home  below  the  lip  of  the  tube.  The  ends 
of  the  U-tube  are  smeared  with  vaseline  and  a  rubber  tube 
slipped  over  each ;  the  end  of  the  culture  tube  being  similarly 

5 


66      METHODS  OF  CULTIVATION  OF  BACTERIA 

treated,  the  free  end  of  one  of  the  rubber  tubes  is  pushed  over  it 
till  the  glass  of  the  U-tube  is  in  contact  with  the  glass  of  the 
culture  tube.  In  the  dry  tube  1  or  2  grammes  of  pyrogallic 
acid  are  placed,  and  the  powder  is  packed  down  with  a  layer  of 
filter  paper.  Ten  or  twenty  cubic  centimetres  of  a  10  per  cent, 
solution  of  sodium  hydrate  are  then  poured  in,  and  the  tube  is 
quickly  connected  up  by  the  rubber  tubing  with  the  other  end 
of  the  U-tube.  In  this  apparatus  the  oxygen  is  absorbed  by  the 
sodium  pyrogallate,  and  the  conditions  for  anaerobic  growth 
are  fulfilled. 

Buchner's  Anaerobic  Tube. — This  may  be  used  either  for 
maintaining  surface  growths  of  anaerobes  or  for  keeping  free 
from  oxygen  sloped  culture  media  which  are  being  used  for 
separating  anaerobes  from  mixtures.  Dry  pyrogallol  is  placed 
in  a  cylindrical  jar  of  diameter  sufficient  to  contain  the  tube 
or  tubes  of  media.  The  tubes  are  v  then  inserted,  potassium 
hydrate  solution  (p.  65)  is  poured  into  the  jar,  and  its  mouth 
quickly  stoppered  with  a  rubber  or  glass  stopper.  The  stopper 
is  made  airtight  by  sealing  with  paraffin.  The  pyrogallol 
absorbs  the  oxygen  in  the  jar,  and  thus  the  cultures  are  kept  in 
oxygen-free  surroundings. 

Growth  in  Tubes  with  Pyrogallol-saturated  Plug. — Sloped 
cultures  can  be  maintained  oxygen-free  as  follows  : — The  medium 
is  placed  in  a  long  test-tube  and  inoculated.  The  plug  of  the 
tube  (which  ought  to  be  rather  tight)  is  pushed  down  into  the 
tube,  and  a  little  dry  pyrogallol  placed  on  the  top  of  it.  A  few 
drops  of  the  potassium  hydrate  solution  are  dropped  on  the 
crystals,  and  a  second  plug  is  inserted  in  the  mouth  of  the  tube. 
This  is  pushed  home,  and  melted  paraffin  run  on  to  the  top  to 
prevent  access  of  outside  air. 

Cultures  of  Anaerobes  in  Liquid  Media. — It  is  necessary  to 
employ  such  in  order  to  obtain  the  toxic  products  of  the  growth 
of  anaerobes.  Glucose  broth  is  most  convenient.  It  is  placed 
either  (1)  in  a  conical  flask  with  a  lateral  opening  and  a  per- 
forated indiarubber  stopper,  through  which  a  bent  glass  tube 
passes,  as  in  Fig.  24,  a,  by  which  hydrogen  may  be  delivered,. 
or  (2)  in  a  conical  flask  with  a  rubber  stopper  furnished  with 
two  holes,  as  in  Fig.  24,  b,  through  a  tube  in  one  of  which 
hydrogen  is  delivered,  while  through  the  tube  in  the  other  the 
gas  escapes.  The  inner  end  of  the  gas  delivery  tube  must  in 
either  case  be  below  the  surface  of  the  liquid ;  the  inner  end  of 
the  lateral  nozzle  in  the  one  case,  and  the  inner  end  of  the 
escape  tube  in  the  other,  must  of  course  be  above  the  surface  of 
the  liquid.  The  single  tube  in  the  one  case  and  the  two  tubes 


CULTURES  OF  ANAEROBES  IN  LIQUID  MEDIA     67 

in  the  other  ought  to  be  partially  drawn  out  in  a  flame  to 
facilitate  subsequent  complete  sealing.  The  ends  of  the  tubes 
through  which  the  gas  is  to  pass  are  previously  protected  by 
pieces  of  cotton  wool  tied  on  them.  It  is  well  previously  to 
place  in  the  tube,  through  which  the  hydrogen  is  to  be  delivered, 
a  little  plug  of  cotton  wool.  The  flask  being  thus  prepared,  it 
is  sterilised  by  methods  B  (2)  or  B  (3).  On  cooling  it  is  ready 
for  inoculation.  In  the  case  of  the  flask  with  the  lateral  nozzle, 
the  cotton-wool  covering  having  been  momentarily  removed,  a 
wire  charged  with  the  organism  is  passed  down  to  the  bouillon. 
In  the  other  kind  of  flask  the  stopper  must  be  removed  for  an 
instant  to  admit  the  wire.  The  flask  is  then  connected  with 


FIG.  24. 

a.  Flask  for   anaerobes  in  liquid  media.     Lateral  nozzle  and  stopper  fitted 
for  hydrogen  supply,    b.  A  stopper  arranged  for  a  flask  without  lateral  nozzle. 

the  hydrogen  apparatus  by  means  of  a  short  piece  of  sterile 
indiarubber  tubing,  and  hydrogen  is  passed  through  for  half  an 
hour.  In  the  case  of  flask  (1),  the  lateral  nozzle  is  plugged 
with  melted  paraffin  and  covered  with  alternate  layers  of  cotton 
wool  and  paraffin,  the  whole  being  tightly  bound  on  with  string. 
The  entrance  tube  is  now  completely  drawn  off  in  the  flame 
before  being  disconnected  from  the  hydrogen  apparatus.  In 
the  case  of  flask  (2),  first  the  exit  tube  and  then  the  entrance 
tube  are  sealed  off  in  the  flame  before  the  flask  is  disconnected 
from  the  hydrogen  apparatus.  It  is  well  in  the  case  of  both 
flasks  to  run  some  melted  paraffin  all  over  the  rubber  stopper. 
Sometimes  much  gas  is  evolved  by  anaerobes,  and  in  dealing 
with  an  organism  where  this  will  occur,  provision  must  be  made 
for  its  escape.  This  is  conveniently  done  by  leading  down  the 


68      METHODS  OF  CULTIVATION  OF  BACTERIA 


exit  tube,  and  letting  the  end  just  dip  into  a  trough  of  mercury 
(Fig.  25),  or  into  mercury  in  a  little  bottle  tied  on  to  the  end 
of  the  exit  tube.  The  pressure  of  gas  within  causes  an  escape 

at  the  mercury  contact, 
which  at  the  same  time  acts 
as  an  efficient  valve.  The 
method  of  culture  in  fluid 
media  is  used  to  obtain  the 
soluble  products  of  such 
anaerobes  as  the  tetanus 
bacillus. 

The  Method  of  Tarozzi.— 
This  observer  has  found  that 
if  small  pieces  of  fresh 
sterile  organs  are  added  to 
ordinary  bouillon,  growth  of 
anaerobes  takes  place  under 
ordinary  atmospheric  con- 
ditions. For  this  purpose, 
portions  of  liver,  spleen,  or 
kidney  are  most  suitable. 
If  after  the  piece  of  tissue  has  been  added  the  medium  is  boiled 
for  a  few  minutes  it  loses  its  property  of  growing  anaerobes, 
but  the  temperature  may  be  raised  for  a  short  time  almost  to 
boiling-point  without  this  occurring.  The  tissue  of  the  organs 
gives  off  something  into  the  medium  which  favours  the  growth 
of  anaerobes,  as  can  be  shown  by  placing  the  tissue  for  some 
time  in  the  medium  and  then  removing  it;  thereafter  the 
medium  is  suitable  for  anaerobic  growth. 

When  it  is  desired  to  grow  anaerobes    on    the  surface  of  a 


FIG.  25. — Flask  arranged  for  culture  of 
anaerobes  which  develop  gas. 

b  is  a  trough  of  mercury  into  which 
exit  tube  dips. 


FIG.  26. — Tubes  for  anaerobic  cultures  on  the  surface  of  solid  media. 

solid  medium  such  as  agar,  tubes  of  the  form  shown  in  Fig.  26, 
a  and  b,  may  be  used.  A  stroke  culture  having  been  made,  the 
air  is  replaced  by  hydrogen  as  just  described,  and  the  tubes  are 


HANGING-DROP  CULTURES  69 

fused  at  the  constrictions.  Such  a  method  is  of  great  value 
when  it  is  required  to  get  the  bacteria  free  from  admixture  of 
medium,  as  in  the  case  of  staining  flagella. 

MISCELLANEOUS  METHODS. 

Hanging-drop  Cultures. — It  is  often  necessary  to  observe 
micro-organisms  alive,  either  to  watch  the  method  and  rate  of 
their  multiplication,  or  to  investigate  whether  or  not  they  are 
motile.  This  is  effected  by  making  hanging-drop  cultures.  The 
method  in  the  form  to  be  described  is  only  suitable  for  aerobes. 
For  this  special  slides  are  necessary.  Two  forms  are  in  use,  and 


FIG.  27. 

A.  Hollow-ground  slide  for  hanging-drop  cultures  shown  in  plan  and  section. 
B.  Another  form  of  slide  for  similar  cultures. 

are  shown  in  Fig.  27.  In  A  there  is  ground  out  on  one  surface 
a  hollow  having  a  diameter  of  about  half  an  inch.  That  shown 
in  B  explains  itself.  The  slide  to  be  used  and  a  cover-glass  are 
sterilised  by  hot  air  in  a  Petri's  dish,  or  simply  by  being  heated 
in  a  Bunsen  and  laid  in  a  sterile  Petri  to  cool.  In  the  case  of 
A,  one  or  other  of  two  manipulation  methods  may  be  employed. 
(1)  If  the  organism  be  growing  in  a  liquid  culture,  a  loop  of 
the  liquid  is  placed  on  the  middle  of  the  under  surface  of  the 
sterile  cover-glass,  which  is  held  in  forceps,  the  points  of  which 
ha\v  IKVH  sterilised  in  a  Bunsen  flame.  If  the  organism  be 
growing  in  a  solid  medium,  a  loopful  of  sterile  bouillon  is 
placed  on  the  cover-glass  in  the  same  position,  and  a  very  small 
quantity  of  the  culture  (picked  up  with  a  platinum  needle)  is 


70     METHODS  OF  CULTIVATION  OF  BACTERIA 

rubbed  up  in  the  bouillon.  The  cover  is  then  carefully  lowered 
over  the  cell  on  the  slide,  the  drop  not  being  allowed  to  touch 
the  wall  or  the  edge  of  the  cell.  The  edge  of  the  cover-glass  is 
covered  with  vaseline,  and  the  preparation  is  then  complete  and 
may  be  placed  under  the  microscope.  If  necessary,  it  may  be  first 
incubated  and  then  examined  on  a  warm  stage.  (2)  The  sterile 
cover-glass  is  placed  on  a  sterile  plate  (an  ordinary  glass  plate 
used  for  plate  cultures  is  convenient).  The  drop  is  then  placed 
on  its  upper  surface,  the  details  being  the  same  as  in  the  last 
case.  The  edge  of  the  cell  in  the  slide  is  then  painted  with 
vaseline,  and  the  slide,  held  with  the  hollow  surface  downwards, 
is  lowered  on  to  the  cover-glass,  to  the  rim  of  which  it  of  course 
adheres.  The  slide  with  the  cover  attached  is  then  quickly 
turned  right  side  up,  and  the  preparation  is  complete. 

In  the  case  of  B,  the  drop  of  fluid  is  placed  on  the  centre  of 
the  table  x.  The  drop  must  be  thick  enough  to  come  in  contact 
with  the  cover-glass  when  the  latter  is  lowered  on  the  slide,  and 
not  large  enough  to  run  over  into  the  surrounding  trench  y. 
The  cover-glass  is  then  lowered  on  to  the  drop,  and  vaseline  is 
painted  along  the  margin  of  the  cover-glass.  The  method  of 
microscopic  examination  is  described  on  page  91. 

The  Counting  of  Colonies.  —  An  approximate  estimate  of  the 
number  of  bacteria  present  in  a  given  amount  of  a  fluid  (say, 
water)  can  be  arrived  at  by  counting  the  number  of  colonies 
which  develop  when  that  amount  is  added  to  a  tube  of  suitable 
medium,  and  the  latter  plated  and  incubated.  An  ordinary 
plate  should  be  used  in  such  a  case,  and  the  medium  poured 

out  in  as  rectangular  a 
shape  as  possible.  For 
the  counting,  an  appa- 
ratus such  as  is  shown 
in;  Fig.  28  is  employed. 
This  consists  of  a  sheet 
of  glass  ruled  into 
squares  as  indicated, 
and  supported  by  its 
corners  on  wooden 
blocks.  The  table  to 

which  these   blocks  are 
FIG.  28.  -Apparatus  for  counting  colonies. 


face.     The  plate-culture 

containing  the  colonies  is  laid  on  the  top  of  the  ruled 
glass.  The  numbers  of  colonies  in,  say,  twenty  of  the  smaller 
squares  are  then  counted,  and  an  average  struck.  The  total 


METHOD  OF  COUNTING  BACTERIA 


number  of  squares  covered  by  the  medium  is  then  taken,  and 
by  a  simple  calculation  the  total  number  of  colonies  present  can 
be  obtained.  Plate-cultures  in  Petri's  dishes  are  sometimes 
employed  for  purposes  of  counting.  The  bottoms  of  such 
dishes  are,  however,  never  flat,  and  the  thickness  of  the  medium 
thus  varies  in  different  parts.  If  these  dishes  are  to  be  used, 
a  circle  of  the  same  size  as  the  dish  can  be  drawn  with  Chinese 
white  on  a  black  card,  the  circumference  divided  into  equal  arcs, 
and  radii  drawn.  The  dish  is  then  laid  on  the  card,  the  number 
of  colonies  in  a  few  of  the  sectors  counted,  and  an  average 
struck  as  before.  In  counting  colonies  it  is  always  best  to  aid 
the  eye  with  a  small  hand-lens. 

Method  of  Counting  Living  Bacteria  in  a  Culture. — This 
is   accomplished   by  putting   into    practice   a  dilution   method 

such  as  that  described  on  p.  58. 
Measured  amounts  of  high  dilutions 
are  plated,  and  the  numbers  of 
colonies  which  subsequently  develop 
are  counted.  In  applying  such  a 
method  it  is  necessary  to  have  pipettes 
capable  of  measuring  small  quantities  of 
fluid.  Those  discharging  '05  and  '1  c.c.  will 
be  found  convenient,  and  such  pipettes  can 
have  subdivisions  which  enable  them  to  be 
used  for  measuring  still  smaller  fractions  of 
a  cubic  centimetre.  Pipettes  of  this  kind 
can  be  obtained  at  the  instrument  makers. 
Wright  has  described  a  method  by  which 
a  pipette  (Fig.  29)  for  measuring  small 
quantities  of  fluid  can  be  made  from  ordinary 
quill  tubing.  The  method  is  as  follows : — 
A  piece  of  quill  tubing  about  15  cm.  long  is 
drawn  out  to  a  capillary  stem.  A  standard 
5  c.mm.  pipette  (such  as  that  of  the  Gower's 
hsemocytometer),  or  the  pipette  described 
later  on  p.  118,  is  filled  with  mercury  and 
the  metal  transferred  to  the  capillary  stem 
and  run  down  to  near  its  extremity;  the 
upper  and  lower  limits  of  the  mercury  are 
marked  with  an  oil  pencil;  the  mercury  is 
then  displaced  up  the  tube  till  its  previously 
distal  end  is  at  the  proximal  of  the  two 
marks,  and  a  third  mark  is  made  at  the  new  position  of 
the  upper  cud  of  the  droplet;  the  manipulation  is  repeated 


250 


225 


20 


15 


10 


2-5 


FlG.  29.— Wright's 
260  c.mm.  pipette 
fitted  with  nipple. 


72      METHODS  OF  CULTIVATION  OF  BACTERIA 

three  more  times,  and  finally  the  tip  of  the  tube  beyond 
the  lowest  mark  is  broken  off.  Thus  on  the  capillary  part 
of  the  pipette  we  have  five  divisions,  each  capable  of 
holding  5  c.mm.  of  fluid.  The  rest  of  the  pipette  is  now 
calibrated  so  as  to  determine  that  part  capable  of  containing 
225  c.mm.  and  250  c.mm.  This  is  done  by  placing  a  rubber 
nipple  on  the  wide  end  of  the  pipette  and  sucking  up  some 
water  tinted  with,  say,  methylene-blue  till  the  25  c.mm.  mark 
is  reached ;  a  small  air-bubble  is  then  allowed  to  enter  the 
pipette,  then  other  25  c.mm.  of  fluid,  then  another  bubble,  and 
so  on  till  nine  volumes  each  of  25  c.mm.  have  been  sucked  up. 
A  mark  is  then  made  on  the  tube  at  the  upper  level  of  this 
amount,  other  25  c.mm.  are  sucked  up,  and  another  mark  made. 
The  fluid  is  expelled,  the  tube  dried,  and  that  part  containing 
the  225  and  250  marks  is  drawn  out  into  an  almost  capillary 
diameter,  the  manipulation  by  which  the  marks  were  originally 
arrived  at  is  repeated,  and  thus  in  the  new  marks  made  a  more 
accurate  calibration  for  these  amounts  is  attained.  In  order  to 
form  a  safety  chamber  a  second  bulb  is  formed  by  drawing  out 
the  tube  a  little  higher  up,  as  in  the  figure,  and  finally  the  upper 
inch  or  two  are  bent  at  right  angles  to  the  calibrated  limb.  In 
doing  this  a  loop  may  be  thrown  on  the  plastic  melted  capillary 
tube  exactly  in  the  way  in  which  a  similar  loop  may  be  thrown 
on  a  piece  of  cord.  With  such  a  pipette  any  required  dilution 
of  a  culture  can  be  made  on  the  principles  already  described. 

The  Bacteriological  Examination  of  the  Blood. — (a)  This 
may  be  done  by  taking  a  small  drop  from  the  skin  surface,  e.g. 
the  lobe  of  the  ear.  The  part  should  be  thoroughly  washed 
with  1-1000  corrosive  sublimate  and  dried  with  sterile  cotton 
wool.  It  is  then  washed  with  absolute  alcohol  to  remove  the 
antiseptic,  drying  being  allowed  to  take  place  by  evaporation. 
A  prick  is  then  made  with  a  sterile  surgical  needle ;  the  drop  of 
blood  is  caught  with  a  sterile  platinum  loop  and  smeared  on  the 
surface  of  agar  or  blood  serum.  Film  preparations  for  micro- 
scopic examination  may  be  made  at  the  same  time.  It  is  rare 
to  obtain  growths  from  the  blood  of  the  human  subject  by  this 
method  (vide  special  chapters),  and  if  colonies  appear  the  pro- 
cedure should  be  repeated  to  exclude  the  possibility  of  accidental 
contamination. 

(b)  A  larger  quantity  of  blood  may  be  obtained  by  puncture 
of  a  vein ;  this  is  the  only  satisfactory  method,  and  should  be 
that  followed  whenever  practicable.  The  skin  over  a  vein  in 
the  forearm  or  on  the  dorsum  of  the  foot  having  been  sterilised, 
the  vein  is  made  turgid  by  pressure,  and  the  needle  of  a  syringe 


EXAMINATION  OF  CEREBRO-SPINAL  FLUID     73 

of  10-15  c.c.  capacity,  carefully  sterilised,  is  then  plunged 
obliquely  through  the  skin  into  the  lumen  of  the  vessel.  Several 
cubic  centimetres  of  blood  can  thus  be  withdrawn  into  the 
syringe.  Some  of  the  blood  (e.g.  1  c.c.)  should  be  added  to 
.small  flasks  containing  50  c.c.  of  bouillon ;  the  rest  may  be 
used  for  smearing  the  surface  of  agar  tubes,  or  may  be  added  to 
melted  agar  at  42°  C.,  which  is  then  plated.  The  flasks,  etc. 
are  then  incubated.  By  this  method  cultures  can  often  be 
obtained  where  the  former  method  fails,  especially  in  severe 
conditions  such  as  ulcerative  endocarditis,  streptococcus  infection, 
etc.  Part  of  the  blood  may  be  incubated  by  itself  for  twenty- 
four  hours,  and  cultures  then  made.  Needless  to  say,  the  in- 
oculations of  media  must  be  done  at  the  bedside,  as  of  course 
the  blood  quickly  coagulates  in  the  syringe.  Coagulation  can 
be  prevented  by  drawing  up  into  the  syringe  before  it  is  used  a 
quantity  of  2  per  cent,  sterile  sodium  citrate  equivalent  to  the 
amount  of  blood  it  is  intended  to  withdraw. 

In  examining  the  blood  of  the  spleen  a  portion  of  the  skin 
over  the  organ  is  sterilised  in  the  same  way,  a  few  drops  are 
withdrawn  from  the  organ  by  a  sterile  hypodermic  syringe,  and 
cultures  made.  (For  microscopic  methods,  vide  p.  94.) 

Bacteriological  Examination  of  the  Cerebro-spinal  Fluid 
—  Lumbar  Puncture.— This  diagnostic  procedure,  which  is 
often  called  for  in  cases  of  meningitis,  can  be  carried  out  with 
a  sterilised  "  antitoxin  needle  "  as  follows  : — The  patient  should 
lie  on  the  right  side,  with  knees  somewhat  drawn  up  and  left 
shoulder  tilted  somewhat  forward,  so  that  the  back  is  fully 
exposed.  The  skin  over  the  lumbar  region  is  then  carefully 
sterilised,  as  above  described,  and  the  hands  of  the  operator 
should  be  similarly  treated.  The  spines  of  the  lumbar  vertebrae 
having  been  counted,  the  left  thumb  or  forefinger  is  pressed 
into  the  space  between  the  third  and  fourth  spines  in  the  middle 
line ;  the  needle  is  then  inserted  about  half  an  inch  to  the 
right  of  the  middle  line  at  this  level  and  pushed  through  the 
tissues,  its  course  being  directed  slightly  inwards  and  upwards, 
till  it  enters  the  subdural  space.  When  this  occurs,  fluid  passes 
along  the  needle,  sometimes  actually  spurting  out,  and  should  be 
received  in  a  sterile  test-tube.  Several  cubic  centimetres  of 
fluid  can  thus  usually  be  obtained,  no  suction  being  required  ; 
thereafter  it  can  be  examined  bacteriological ly  by  the  usual 
methods.  The  depth  of  the  subdural  space  from  the  surface 
varies  from  a  little  over  an  inch  in  children  to  3  inches,  or 
even  more,  in  adults — the  length  of  the  needle  must  be  suited 
accordingly.  In  making  the  puncture  it  is  convenient  to  have 


74      METHODS  OF  CULTIVATION  OF  BACTERIA 

either  a  sterile  syringe  attached,  or  to  have  the  thick  end  of 
the  needle  covered  with  a  pad  of  sterile  wool,  which  is  of  course 
removed  at  once  when  the  fluid  begins  to  flow.  It  is  advisable 
to  use  the  platinum  needles  which  are  specially  made  for  the 
purpose,  as  a  sudden  movement  of  the  patient  may  snap  an 
ordinary  steel  needle. 

The  Bacteriological  Examination  of  Urine. — In  such  an 
examination  care  must  be  taken  to  prevent  the  contamination 
of  the  urine  by  extraneous  organisms.  In  the  male,  specimens 
withdrawn  by  a  sterile  catheter  into  a  sterile  vessel  are  pre- 
ferable, but  it  is  often  sufficient  to  wash  thoroughly  the  glans 
penis  and  the  meatus  with  1-1000  corrosive  sublimate — the 
lips  of  the  meatus  being  everted  for  more  thorough  cleansing ; 
the  urine  is  then  passed  into  a  series  of  sterile  flasks,  the  first 
of  which  is  rejected  in  case  contamination  has  occurred.  In 
the  female,  after  similar  precautions  as  regards  external 
cleansing,  the  catheter  must  be  used.  The  latter  must  be 
boiled  for  half  an  hour,  and  anointed  with  olive  oil  sterilised 
by  half  an  hour's  exposure  in  a  plugged  flask  to  a  temperature 
of  120°  C.  Here,  again,  it  is  well  to  reject  the  urine  first 
passed.  It  is  often  advisable  to  allow  the  urine  to  stand  in  a 
cool  place  for  some  hours,  to  then  withdraw  the  lower  portion 
with  a  sterile  pipette,  to  centrifugalise  this,  and  to  use  the 
urine  in  the  lower  parts  of  the  centrifuge  tubes  for  microscopic 
examination  or  culture. 

Filtration  of  Cultures. — For  many  purposes  it  is  necessary 
to  filter  all  the  organisms  from  fluids  in  which  they  may  have 
been  growing.  This  is  done  especially  in  obtaining  the  soluble 
toxic  products  of  bacteria.  The  only  filter  capable  of  keeping 
back  such  minute  bodies  as  bacteria  is  that  formed  from  a  tube 
of  unglazed  earthenware  as  introduced  by  Chamberland.  The 
efficiency  of  such  a  filter  depends-  on  the  fineness  of  the  grain 
of  the  clay  from  which  it  is  made ;  the  finest  is  the  Kitasato 
filter  and  the  Chamberland  "  B  "  pattern ;  the  next  finest  is 
the  Chamberland  "  F  "  pattern,  which  is  quite  good  enough  for 
ordinary  work.  There  are  several  filters,  differing  slightly  in 
detail,  all  possessing  the  common  principle.  Sometimes  the 
fluid  is  forced  through  the  porcelain  tube.  In  one  form  the 
filter  consists  practically  of  an  ordinary  tap  screwed  into 
the  top  of  a  porcelain  tube.  Through  the  latter  the  fluid  is 
forced,  and  passes  into  a  chamber  formed  by  a  metal  cylinder 
which  surrounds  the  porcelain  tube.  The  fluid  escapes  by  an 
aperture  at  the  bottom.  Such  a  filter  is  very  suitable  for 
domestic  use,  or  for  use  in  surgical  operating-theatres.  As 


FILTRATION  OF  CULTURES 


75 


considerable  pressure  is  necessary,  it  is  evident  it  must  be  put 
on  a  pipe  leading  directly  from  the  main.  Sometimes,  when 
fluids  to  be  filtered  are 
very  albuminous,  they 
are  forced  through  a 
porcelain  cylinder  by 
compressed  carbonic 
acid  gas.  The  filtra- 
tion of  albuminous 
fluids  may  sometimes 
be  facilitated  by  keep- 
ing them  near  blood- 
heat  during  the  pro- 
cess. For  ordinary 
bacteriological  work, 
filters  of  various  kinds 
are  in  the  market 
(such  as  those  of  Klein 
and  others),  but  the 
most  generally  con- 
venient is  that  in 
which  the  fluid  is  FIG.  30.— Geissler's  vacuum  pump  arranged  with 
sucked  through  the  manometer  for  filtering  cultures.  (The  tap 
i  •  r  i  and  pump  are  intentionally  drawn  to  a  larger 

porcelain    by   exhaust-      ^  &£  the  manometer  board   to   show 
ing     the    air    in    the      details.) 
receptacle   into    which 

it  is  to  flow.  This  is  conveniently  done  by  means  of  a 
Geissler's  water-exhaust  pump  (Fig.  30,  g\  which  must  be 
fixed  to  a  tap  leading  directly  from  the  main.  The  connection 

with  the  tap  must  be  effected  by 
means  of  a  piece  of  thick-walled 
rubber-tubing  as  short  as  possible, 
wired  on  to  tap  and  pump,  and 
firmly  lashed  externally  with  many 
turns  of  strong  tape.  Before  lashing 
with  the  tape  the  tube  may  be 
strengthened  by  fixing  round  it 
with  rubber  solution  strips  of  the 
rubbered  canvas  used  for  mending 
punctures  in  the  outer  case  of  a 
bicycle  tyre.  A  manometer  tube 
(b)  and  a  receptacle  (c)  (the  latter 

to  catch  any  back  flow  of  water  from  the  pump  if   the  filter 
breaks)  are  intercepted  between  the  filter  and  the 


FIG.  31.— Chamberland's  candle 
and  Hask  arranged  for  filtra- 
tion. 


76      METHODS  OF  CULTIVATION  OF  BACTERIA 

pump.  These  are  usually  arranged  on  a  board  a,  as  in  Fig.  30. 
Between  the  tube  /  and  the  pump  g,  and  between  the  tube  d 
and  the  filter,  it  is  convenient  to  insert  lengths  of  flexible 
lead-tubing  connected  up  at  each  end  with  short,  stout-walled 
rubber-tubing. 

Filters  are  arranged  in  various  ways,  (a)  An  apparatus  is 
arranged  as  in  Fig.  31.  The  fluid  to  be  filtered  is  placed  in 
the  cylindrical  vessel  a.  Into  this  a  "  candle  "  or  "  bougie  " 
of  porcelain  dips.  From  the  upper  end  of  the  bougie  a  glass 
tube  with  thick  rubber  connections,  as  in  Fig.  31,  proceeds  to 
flask  b,  and  passes  through  one  of  the  two  perforations  with 
which  the  rubber  stopper  of  the  flask  is  furnished.  Through 


FIG.  32. — Cliamberland's  bougie 
arranged  with  lamp  funnel  for 
filtering  a  small  quantity  of 
riuid. 


FIG.  33.— Bougie  inserted 
through  rubber  stopper 
for  same  purpose  as  in 
Fig.  32. 


the  other  opening  a  similar  tube  proceeds  to  the  exhaust- 
pump.  When  the  latter  is  put  into  action  the  fluid  is  sucked 
through  the  porcelain  and  passes  over  into  flask  b.  This 
apparatus  is  very  good,  but  not  suitable  for  small  quantities  of 
fluid. 

(b)  A  very  good  apparatus  can  be  arranged  with  a  lamp 
funnel  and  the  porcelain  bougie.  These  may  be  fitted  up  in 
two  ways.  (1)  An  indiarubber  washer  is  placed  round  the 
bougie  c  at  its  glazed  end  (vide  Fig.  32).  On  this  the  narrow 
end  of  the  funnel  d,  which  must,  of  course,  be  of  an  appropriate 
size,  rests.  A  broad  band  of  sheet  rubber  is  then  wrapped 
round  the  lower  end  of  the  funnel,  and  the  projecting  part  of 
the  bougie.  It  is  firmly  wired  to  the  funnel  above  and  to  the 


FILTRATION  OF  CULTURES 


77 


bougie  below.  The  extreme  point  of  the  latter  is  left  exposed, 
and  the  whole  apparatus,  being  supported  on  a  stand,  is  con- 
nected by  a  glass  tube  with  the  lateral  tube  of  the  flask  b ;  the 
tube  a  is  connected  with  the  exhaust-pump.  The  fluid  to  be 
filtered  is  placed  between  the  funnel  and  the  bougie  in  the 
space  e,  and  is  sucked  through  into  the  flask  b.  The  efficiency 
of  such  a  filter,  especially  when  small  amounts  of  fluid  are  being 
dealt  with,  is  much  increased  if  when  the  level  of  the  fluid  falls 
below  the  upper  end  of  the  candle  a  closely  fitting  test-tube  is 
slipped  over  the  latter.  By  this  device  the  leakage  of  air 
through  the  exposed  part  of  the  candle  is  prevented.  There 
are  now  in  the  market  candles  with  glass  sheaths  cemented  into 
a  nickle-plated  fitting  from  the  lower  part  of  which  a  metal 
tube  emerges;  the  latter 
can  be  passed  through  a 
rubber  stopper  into  a  filter 
tlask.  (2)  This  modifica- 
tion is  shown  in  Fig.  33. 
Into  the  narrow  part  of 
the  funnel  an  indiarubber 
bung  is  fitted,  with  a  per- 
foration in  it  sufficiently 
large  to  receive  the  candle, 
which  it  should  grasp 
tightly. 

(o)  Muencke's  modifica- 
tion of  the  Chamberland 
filter  is  seen  in  Fig.  34. 
It  consists  of  a  thick- 
\\alird  tlask  a,  the  lower 

part  conical,  the  upper  cylindrical,  with  a  strong  flange  on  the 
lip.  There  are  two  lateral  tubes,  one  horizontal  to  connect  with 
exhaust-pi] >e,  and  one  sloping,  by  which  the  contents  may  be 
j  toured  out.  Passing  into  the  upper  cylindrical  part  of  the 
tlask  is  a  hollow  porcelain  cylinder  6,  of  less  diameter  than  the 
cylindrical  part  of  flask  a.  It  is  closed  below,  open  above,  and 
rests  by  a  projecting  rim  on  the  flange  of  the  flask,  an  asbestos 
\\asher,  c,  being  interposed.  The  fluid  to  be  filtered  is  placed 
in  the  porcelain  cylinder,  and  the  whole  top  covered,  as  shown 
at  /,  with  an  indiarubber  cap  with  a  central  perforation ;  the 
tube  d  is  connected  with  the  exhaust-pump,  and  the  tube  e 
plugged  with  a  rubber  stopper.  For  filtering  small  quantities 
of  fluid  the  apparatus  shown  in  Fig.  35  may  be  used.  It 
consists  of  a  small  Chamberland  bougie  fitted  by  a  rubber  tube 


Fi({.  34. — Muencke's  modification  of 
Chamberland's  filter. 


78     METHODS  OF  CULTIVATION  OF  BACTERIA 


to  a  funnel,  the  stem  of  which  has  been  passed  through  a  rubber 
cork;  this  cork  fits  into  a  conical  flask  with  side  arm  for 
connection  with  exhaust. 

Before  any  one  of  the  above  apparatus  is  used  it  ought  to 
be  connected  up  as  far  as  possible  and  sterilised  in  the  Koch's 
steriliser.  The  ends  of  any  important 
unconnected  parts  ought  to  have  pieces  of 
cotton  wool  tied  over  them.  After  use 
the  bougie  is  to  be  sterilised  in  the  auto- 
clave, and  after  being  dried  is  to  be  passed 
carefully  through  a  Bunsen  name  to  burn 
off  all  organic  matter.  If  the  latter  is 
allowed  to  accumulate,  the  pores  become 
filled  up. 

The  success  of  filtration  must  be  tested 
by  inoculating  tubes  of  media  from  the 
filtrate,  and  observing  if  growth  takes 
place,  as  there  may  be  minute  perforations 
in  the  candles  sufficiently  large  to  allow 
bacteria  to  pass  through.  Filtered  fluids 
keep  for  a  long  time  if  the  openings  of 
the  glass  vessels  in  which  they  are  placed 
are  kept  thoroughly  closed,  and  if  these 
vessels  be  kept  in  a  cool  place  in  the  dark. 
A  layer  of  sterile  toluol  about  half  an  inch 
thick  ought  to  be  run  on  to  the  top  of  the 
filtered  fluid  to  protect  the  latter  from  the 
atmospheric  oxygen. 

Instead  of  being  filtered  off,  the  bacteria 
may  be  killed  by  various  antiseptics, 
chiefly  volatile  oils,  such  as  oil  of  mustard 

(Roux).  These  oils  are  stated  to  have  no  injurious  effect  on  the 
chemical  substances  in  the  fluid,  and  they  may  be  subsequently 
removed  by  evaporation.  It  is  not  practicable  to  kill  the. 
bacteria  by  heat  when  their  soluble  products  are  to  be  studied, 
as  many  of  the  latter  are  destroyed  by  a  lower  temperature  than 
is  required  to  kill  the  bacteria  themselves. 

Bacteria  can  be  almost  entirely  removed  from  fluid  cultures 
by  spinning  the  latter  in  a  centrifuge  of  very  high  speed  (e.g. 
C.  J.  Martin's  turbine  centrifuge),  and  this  method  is  sometimes 
adopted  in  practice. 

The  Observation  of  Bacterial  Fermentation  of  Sugars,  etc. 
—The  capacity  of  certain  species  of  bacteria  to  originate  fermenta- 
tions in  sugars  constitutes  an  important  biological  factor.  It 


FIG.  35.  —  Flask  for 
filtering  small  quanti- 
ties of  fluid. 


BACTERIAL  FERMENTATION  OF  SUGARS       79 

is  well  to  consider  this  factor  in  relation  to  the  chemical  con- 
stitution of  the  sugars.  These  bodies  are  now  known  to  be  (to 
use  the  definition  of  Holleman)  aldehyde  or  ketoue  alcohols 
containing  one  or  more  hydroxyl  groups,  one  of  which  is  directly 
linked  to  a  carbon  atom  in  union  with  car  bony  1.  The  group 
characteristic  of  a  sugar  is  thus  —  CHOH  -  CO  - .  The  sugars 
are  divided  into  monosaccharides  or  monoses,  disaccharides 
(dioses),  and  polysaccharides  (polyoses).  The  members  of  the 
last  two  groups  may  be  looked  on  as  derived  from  the  combina- 
tion of  two  or  more  molecules  of  a  monosaccharide  with  the 
elimination  of  water  (e.g.  2C6Hr2O6  =  C1?H.22OU  +  H?O). 

Monosaccharides.  —  These  are  classified  according  to  the 
number  of  C  atoms  they  contain.  The  pentoses  ordinarily  used 
are  arabinose  (obtained  from  gum  arabic),  xylose  (from  wood), 
and  rhamnose  (which  is  really  a  methylpentose).  Among  the 
hexoses  are  glucose  (dextrose)  with  dextro-rotatory  properties. 
Glucose  is  an  aldehyde  alcohol  (aldose).  In  fruit  there  is  also 
a  ketone  alcohol  (ketose)  called  fructose,  which  from  its  laevo- 
rotatory  properties  is  also  known  as  laevulose.  Other  hexosee 
are  mannose  (from  the  vegetable  ivory  nut)  and  galactose  (a 
hydrolytic  derivative  of  lactose). 

Disaccharides  (C12H2.2On). — The  ordinary  members  of  this 
group  are  maltose  (derived  from  starch),  lactose,  and  cane  sugar 
(sucrose,  saccharose). 

Polysaccharides. — Examples  are  starch,  raffinose,  inulin  (from 
dahlia  roots),  dextrin,  arabin,  glycogen,  cellulose. 

If  we  consider  sugars  generally  from  the  point  of  view  of 
the  capacity  of  yeast  to  originate  alcoholic  fermentation  in  them, 
we  may  say  that  the  simpler  the  constitution  of  the  sugar  the 
more  easily  is  it  fermented.  Thus  the  monosaccharides  are 
more  easily  acted  on  by  yeast  than  the  di-  or  poly-saccharides. 
Usually  an  independent  process  resulting  in  the  splitting  of  the 
higher  into  the  lower  is  preliminary  to  the  alcoholic  fermentation. 
Thus  yeast  first  inverts  cane  sugar  into  glucose  and  fructose,  and 
then  acts  on  these  products.  From  what  is  known  it  is  probable 
that  similar  facts  hold  with  regard  to  the  action  of  bacteria. 

Besides  sugars,  other  alcohols  with  large  molecules  may  be 
broken  down  by  bacterial  action,  and  these  bodies  have  been 
used  for  differentiating  the  properties  of  allied  bacteria.  Among 
these  substances  may  be  mentioned  the  trihydric  alcohol  glycerol 
(glycerin),  the  tetrahydric  erythritol  and  the  hexahydric  dulcitol 
(dulcite),  mannitol  (mannite),  and  sorbitol  (sorbite). 

Similarly  certain  glucosides,  such  as  salicin,  coniferin,  etc., 
have  been  used  for  testing  the  fermentative  properties  of 


80     METHODS  OF  CULTIVATION  OF  BACTERIA 

bacteria.  Other  substances  allied  to  sugars  (e.g.  inosite)  have 
also  been  used. 

The  end  products  of  bacterial  fermentations  may  be  various. 
They  differ  according  to  the  sugar  employed  and  according  to 
the  species  of  bacterium  under  observation,  and  frequently  a 
species  which  will  ferment  one  sugar  has  no  effect  on  another. 
The  substances  finally  produced,  speaking  roughly,  may  be 
alcohols,  acids,  or  gaseous  bodies  (chiefly  carbon  dioxide, 
hydrogen,  and  methane).  For  the  estimation  of  the  first  groups 
complicated  chemical  procedure  may  be  necessary.  The  tests 
usually  employed  for  the  detection  of  ordinary  fermentative 
processes  depend  on  two  kinds  of  changes,  namely,  (a)  the  evolution 
of  gases  and  (6)  the  formation  of  acids.  Generally  speaking,  we 
may  say  that  such  tests  are  reliable,  and  the  methods  to  be 
pursued  are  simple.  Besides  such  gases  as  those  named,  some 
organisms  give  rise  to  sulphuretted  hydrogen  by  breaking  up  the 
proteid.  The  formation  of  this  gas  can  be  detected  by  the 
blackening  of  lead  acetate  when  it  is  added  to  the  gas-containing 
medium. 

In  testing  the  effect  of  a  bacterium  on  a  given  sugar  it  is 
essential  that  this  sugar  alone  be  present;  the  basis  of  the 
medium  ought  therefore  to  be  either  peptone  solution  (vide  p.  39) 
or  a  dextrose-free  bouillon  (vide  infra).  The  sugar  or  other 
substance  is  added  in  the  proportion  of  from  a  half  to  one 
per  cent.,  and  care  is  taken  not  to  overheat  during  sterilisation. 

It  is  preferable  that  the  addition  should  Le  made  in  the  form  of  a  sterile 
solution.  If  the  sugar  in  solid  form  be  placed  in  the  bouillon  and  this 
then  sterilised,  there  is  danger  that  chemical  changes  may  take  place 
in  the  sugar,  in  consequence  of  its  being  heated  in  the  presence  of 
substances  (such  as  alkalies)  which  may  act  deleteriously  upon  it ;  in  any 
case  sterilisation  should  not  be  at  a  temperature  above  100°  C. 

To  obtain  a  "  dextrose- free"  bouillon  it  is  usual  to  inoculate  ordinary 
bouillon  with  some  organism,  such  as  b.  coli,  which  is  known  to  ferment 
dextrose,  and  allow  it  to  act  for  forty-eight  hours.  The  bouillon  is 
then  filtered  and  re-sterilised.  A  sample  is  tested  for  another  period 
of  forty-eight  hours  with  b.  coli,  to  make  certain  that  all  the  dextrose 
has  been  removed.  If  no  fresh  gas-formation  is  observed,  then  to  the 
remainder  of  the  bouillon  the  sugar  to  be  investigated  may  be  added. 

For  the  observation  of  gas-formation  either  of  the  following 
methods  may  be  employed  : — 

(1)  Durham's  Tubes  (Fig.  36,  b).— The  plug  of  a  tube  which 
contains  about  one-third  more  than  usual  of  a  liquid  medium  is 
removed,  and  a  small  test-tube  is  slipped  into  the  latter,  mouth 
downwards.  The  plug  is  replaced  and  the  tube  sterilised  thrice 
for  ten  minutes  at  100°  C.  The  air  remaining  in  the  smaller 


BACTERIAL  FERMENTATION  OF  SUGARS       81 

tube  is  thereby  expelled.  The  tube  is  then  inoculated  with  the 
bacterium  to  be  tested.  Any  gas  developed  collects  in  the  upper 
part  of  the  inner  tube.  As  some  of  the  sugars  now  used  for 
fermentation  tests  are  rather  expensive,  it  is  well  to  arrange  the 
Durham  apparatus  with  very  small  tubes ;  with  these  a  satis- 
factory result  can  be  obtained  with  only  1  c.c.  of  medium. 

(2)  T/te  Fermentation  Tube  (Fig.  36,  c). — This  consists  of  a 
tube  of  the  form  shown,  and  the  figure  also  indicates  the  extent 
to  which  it  ought  to  be  filled.  It  is  inoculated  in  the  bend  with 
the  gas-forming  organism,  and  when  growth  occurs  the  gas 


FIG.  36. — Tubes  for  demonstrating  gas-fonaatiou  by  bacteria. 

",  tube  with  "shake"  culture. 

b,  Durham's  fermentation  tube. 

c,  ordinary  form  of  fermentation  tube. 

collects  in  the  upper  part  of  the  closed  limit,  the  medium  being 
displaced  into  the  bulb. 

For  the  observation  of  the  effect  of  an  organism  on  glucose, 
the  following  method  may  be  employed  : — 

Gelatin  Shake  Cultures  (Fig.  36,  a). — The  gelatin  in  the  tube 
is  melted  as  for  making  plates  ;  while  liquid  it  is  inoculated 
witli  the  growth  to  be  observed,  and  shaken  to  distribute  the 
organisms  throughout  the  jelly.  It  is  then  allowed  to  solidify, 
;tn  1  i-  set  a>ide  at  a  suitable  temperature.  If  the  bacterium  used 
is  a  gas-forming  one,  then,  as  growth  occurs,  little  bubbles 
appear  round  the  colonies. 
6 


82      METHODS  OF  CULTIVATION  OF  BACTERIA 

In  this  method  the  gas-formation  results  from  fermentation 
of  the  glucose  naturally  present  in  the  medium  from  transforma- 
tion of  the  carbo-hydrates  of  muscle.  The  amount  of  glucose 
naturally  present,  however,  varies  much,  and  therefore  glucose 
should  be  added  to  the  medium  if  the  effects  on  this  sugar  are 
to  be  observed  with  certainty.  The  shake  culture  method  may 
be  utilised  for  observing  fermentation  in  other  sugars  by  adding 
to  peptone  solution  containing  the  sugar  10  to  15  per  cent,  of 
gelatin. 

The  development  of  an  acid  reaction  is  demonstrated  by  the 
addition  of  an  indicator  to  the  medium,  litmus  being  generally 
used.  The  details  of  the  composition  of  such  media  have  already 
been  given.  In  Hiss's  serum  water  media  the  production  of 
acid  also  leads  to  coagulation  of  the  medium.  Sometimes  acid 
is  formed  very  slowly  from  sugars,  so  that  it  is  well  to  keep  the 
cultures  under  observation  for  several  days. 

Acid  and  gas-formation  may  be  simultaneously  tested  for,  by 
placing  the  fluid  medium  containing  the  indicator  in  Durham's 
tubes. 

In  all  tests  in  which  sugars  are  used,  a  control  uninoculated 
tube  ought  to  be  incubated  along  with  the  bacterial  cultures,  as 
changes  in  reaction  sometimes  spontaneously  occur  in  media 
containing  unstable  sugars. 

The  capacity  of  an  organism  to  produce  acid  may  be  measured 
by  taking  a  standard  amount  of  a  fluid  medium  and  allowing 
growth  to  take  place  for  a  standard  time,  and  then  adding  an 
amount  of,  say,  decinormal  soda  solution  sufficient  to  bring  the 
litmus  back  to  the  tint  of  the  original  medium. 

The  Observation  of  Indol-fonnation  by  Bacteria. — The 
formation  of  indol  from  albumin  by  a  bacterium  sometimes 
constitutes  an  important  specific  characteristic.  To  observe 
indol  production  the  bacterium  is  grown,  preferably  at  incubation 
temperature,  in  a  fluid  medium  containing  peptone.  The  latter 
may  either  be  sugar-free  bouillon  or  preferably  peptone  solution 
(see  p.  39).  Any  medium  containing  sugars  must  be  avoided,,  as 
the  presence  of  these  substances  may  inhibit  the  production  of 
indol.  Two  methods  are  in  use  for  the  detection  of  this  body. 

(1)  The  Nitroso-indol  Method. — Indol  is  here  recognised  by 
the  fact  that  when  it  is  acted  on  by  nitric  acid  in  the  presence  of 
nitrites,  a  nitroso-indol  compound  is  produced,  which  has  a  rosy 
red  colour.  Some  bacteria  (e.g.  the  cholera  vibrio)  produce 
nitrites  as  well  as  indol,  but  usually  in  making  the  test  (e.g.  in 
the  case  of  b.  coli)  the  nitrites  must  be  added.  This  is  effected 
by  adding  to  an  ordinary  tube  of  medium  1  c.c.  of  a  '02  per 


INDOL-FORMATION  BY  BACTERIA  83 

cent,  solution  of  potassium  nitrite,  and  testing  with  pure  nitric 
or  sulphuric  acid.  In  any  case  only  a  drop  of  the  acid  need  be 
added  to,  say,  10  c.c.  of  medium.  If  no  result  be  obtained  at 
once  it  is  well  to  allow  the  tube  to  stand  for  an  hour,  as  some- 
times the  reaction  is  very  slowly  produced.  In  many  instances 
incubation  at  37°  C.  for  several  days  may  be  necessary  before 
tlir  presence  of  indol  is  demonstrable.  The  amount  of  indol 
produced  by  a  bacterium  seems  to  vary  very  much  with  certain 
unknown  qualities  of  the  peptone.  It  is  well,  therefore,  to  test 
a  series  of  peptones  with  an  organism  (such  as  the  b.  coli) 
known  to  produce  indol,  and,  noting  the  sample  with  which  the 
best  reaction  is  obtained,  to  reserve  it  for  making  media  to  be 
used  for  the  detection  of  this  product.  This  method  has  for 
long  been  felt  not  to  be  satisfactory,  and  the  following  at  present 
bids  fair  to  replace  it : — 

(2)  Ehrlictis  Rosindol  Reaction-: — The  adaptation  of  this  to 
bacteriological  purposes  was  brought  forward  by  Bohme  in  1906. 
For  ease  of  application  and  delicacy  of  effect  the  reaction 
possesses  great  advantages.  It  depends  on  the  fact  that 
paradimethylamidobenzaldehyde  unites  with  indol  to  form  a 
rosindol  body  whose  colour  is  readily  developed,  especially  in 
presence  of  an  oxidising  substance  such  as  potassium  per- 
sulphate (K2SoO8).  Two  solutions  are  required  : — 

(1)  Paradimethylamidobenzaldehyde  (Grubler)  4  grms. 
Absolute  alcohol  (96  per  cent.)  .  .  380  c.c. 
Concentrated  hydrochloric  acid  .  .  80  c.c. 

(•2)  Potassium  persulphate      .      Saturated  watery  solution. 

To  a  10  c.c.  bouillon  culture  of  the  organism  add  5  c.c.  of  (1) 
and  then  5  c.c.  of  (2),  and  shake  well  (according  to  MacConkey 
1  c.c.  of  each  solution  is  sufficient) ;  if  indol  be  present  a  rose- 
red  colour  will  appear  in  a  few  minutes.  Sometimes  the  rose 
colour  appears  on  the  addition  of  solution  (1),  and  the  addition 
of  a  special  oxidising  agent  is  unnecessary.  The  rosindol  com- 
pound can  be  separated  from  the  culture  by  shaking  the  latter 
up  with  amyl  alcohol,  and  MacConkey  recommends  that  this 
should  be  done  in  cases  of  a  doubtful  reaction,  as  sometimes 
when  a  faint  pink  colour  appears  in  the  culture  tube  the 
extracting  alcohol  remains  colourless,  showing  that  no  real 
reaction  has  occurred.  Marshall  has  pointed  out  that  by  means 
of  the  reaction  a  quantitative  estimate  of  the  amount  of  indol 
formation  can  be  obtained.  To  do  this  a  large  culture,  say 
100  c.c.,  is  distilled,  and  the  colour  obtained  by  applying  the 


84      METHODS  OF  CULTIVATION  OF  BACTERIA 

test  to  the  distillate  in  a  Nessler's  tube  is  matched  against  that 
obtained  with  different  amounts  of  a  standard  solution  of  indol 
(prepared  by  dissolving  1  gr.  indol  in  5  c.c.  absolute  alcohol,  and 
making  up  to  500  c.c.  with  distilled  "water). 

There  is  no  doubt  that  the  Ehrlich  test  is  from  five  to  ten 
times  more  delicate  than  the  ordinary  nitroso-indol  reaction,  and 
it  is  of  especial  value  in  dealing  with  organisms  of  the  coli- 
typhoid  group.  With  strains  of  b.  coli  it  can  often  be  obtained 
in  from  twenty-four  to  forty-eight  hours,  but  in  the  case  of  a  nega- 
tive result  a  culture  of  from  six  to  seven  days  ought  to  be  used. 
The  reaction  is  also  obtainable  with  the  cholera  vibrio,  but  further 
investigation  is  here  necessary,  as  Marshall  states  that  under 
certain  circumstances  the  nitrites  formed  by  this  bacterium  may 
have  an  inhibitory  effect  on  the  production  of  the  rose  colour. 

The  Drying  of  Substances  in  vacuo. — As  many  substances, 
for  example  toxins  and  antitoxins,  with  which  bacteriology  is 
concerned  would  be  destroyed  by  drying  with  heat  as  is  done  in 
ordinary  chemical  work,  it  is  necessary  to  remove  the  water  at 
the  ordinary  room  temperature.  This  is  most  quickly  effected 
by  drying  in  vacuo  in  the  presence  of  some  substance  such  as 
strong  sulphuric  acid,  which  readily  takes  up  water  vapour.  The 
vacuum  produced  by  a  water-pump  is  here  not  available,  as  in 
such  a  vacuum  there  must  always  be  water  vapour  present.  An 
air-pump  is  therefore  to  be  employed.  Here  we  have  found  the 
Geryk  pump  most  efficient,  and  it  has  this  further  advantage, 
that  its  internal  parts  are  lubricated  with  an  oil  of  very  low 
vapour  density,  so  that  almost  a  perfect  vacuum  is  obtainable. 
The  apparatus  is  shown  in  Fig.  37.  The  vacuum  chamber 
consists  of  a  bell-jar  set  on  a  brass  plate.  A  perforation  in  the 
centre  of  the  latter  leads  into  the  pipe  a,  W7hich  can  be  connected 
by  strong- walled  rubber-tubing  with  the  air-pump,  and  wThich 
can  be  cut  off  from  the  latter  by  a  stop-cock  I.  In  using  the 
apparatus  the  substance  to  be  dried  is  poured  out  in  flat  dishes 
(one-half  of  a  Petri  capsule  does  very  well),  and  these  are  stacked 
alternately  with  similar  dishes  of  strong  sulphuric  acid  on  a 
stand  which  rests  on  the  brass  plate.  The  edge  of  the  bell-jar 
is  well  luted  with  unguentum  resinse  and  placed  in  position  and 
the  chamber  exhausted.  In  a  few  hours,  if,  as  is  always  advis- 
able, each  dish  have  contained  only  a  thin  layer  of  fluid,  the 
drying  will  be  complete.  The  vacuum  is  then  broken  by 
admitting  air  very  slowly  through  a  by-pass  c,  and  the  bell-jar 
is  removed.  In  such  an  apparatus  it  is  always  advisable,  as  is 
shown  in  the  figure,  to  have  interposed  between  the  pump  and 
the  vacuum  chamber  a  Wolff's  bottle  containing  sulphuric  acid, 


STORING  AND  INCUBATION  OF  CULTURES      85 

This  protects  the  oil  of  the  pump  from  contamination  with 
water  vapour.  Whenever  the  vacuum  is  produced,  the  rubber- 
tube  should  be  at  once  disconnected  from  a,  the  cock  b  being 
shut.  It  is  advisable  when  the  apparatus  is  exhausted  to  cover 
the  vacuum  chamber  and  the  Wolff's  bottle  with  wire  guards 
covered  with  strong  cloth,  in  case,  under  the  external  pressure, 
the  glass  vessels  give  way.  The  connecting  and  disconnecting 
of  rubber-tubing  of  sufficient  thickness  to  withstand  collapse 
when  exhausted  is  difficult.  Ordinary  stout  rubber-tubing  can 
be  used  if  through  it  there  is  passed  a. length  of  narrow7  flexible 


FIG.  37. — Geryk  air-pump  for  drying  in  varno. 

metal-tubing,  the  ends  of  which  project  beyond  the  rubber-tubing 
so  as  to  enter  the  parts  of  the  apparatus  to  which  the  latter  is 
fitted. 

The  Storing  and  Incubation  of  Cultures. — Gelatin  cultures 
must  be  grown  at  a  temperature  below  their  melting-point,  i.e. 
for  10  per  cent,  gelatin,  below  22°  C.  They  are  usually  kept  in 
ordinary  rooms,  which  vary,  of  course,  in  temperature  at  different 
times,  but  which  have  usually  a  range  of  from  about  12°  C.  to 
18°  C.  Agar  and  serum  media  are  employed  to  grow  bacteria 
at  a  higher  temperature,  corresponding  to  that  at  which 
the  organisms  grow  best,  usually  37°  C.  in  the  case  of  patho- 
genic organisms.  For  the  purpose  of  maintaining  a  uniform 
temperature  incubators  are  used.  These  vary  much  in  the 


86      METHODS  OF  CULTIVATION  OF  BACTERIA 

details  of  their  structure,   but   all  consist  of  a  chamber    with 
double  walls  between  which  some  fluid  (water  or  glycerin  and 
wrater)  is  placed,  which,  when  raised  to  a  certain  temperature, 
ensures    a    fairly    constant    distribution    of 
&  the  heat  round  the  chamber.      The  latter 

is  also  furnished  with  double  doors,  the 
inner  being  usually  of  glass.  Heat  is  sup- 
plied from  a  burner  fixed  below.  These 
burners  vary  much  in  design.  Sometimes 
a  mechanism  devised  in  Koch's  laboratory 
is  affixed,  which  automatically  turns  off  the 
gas  if  the  light  be  accidentally  extinguished. 
Between  the  tap  supplying  the  gas,  and  the 
burner,  is  interposed  a  gas  regulator.  Such 
regulators  vary  in  design,  but  for  ordinary 
chambers  which  require  to  be  kept  at  a 
constant  temperature,  Reichert's  is  as  good 
and  simple  as  any,  and  is  not  expensive. 
It  is  shown  in  Fig.  38. 

It  consists  of  a  long  tube  /  closed  at  the  lower 
end,  open  at  the  upper,  and  furnished  with  two 
lateral  tubes.     The  lower  part  is  filled  with  mer- 
cury up  to  a  point  above  tlie  level  of  the  lower 
lateral  tube.     The  end  of  the  latter  is  closed  by 
a  brass  cap  through  which  a  screw  d  passes,  the 
FIG.  38. — Reichert's       inner  end  of  which  lies  free  in  the  mercury.     The 
gas  regulator.  height  of  the  latter   in   the  perpendicular  tube 

can  thus  be  varied  by  increasing  or  decreasing  the 

capacity  of  the  lateral  tube  by  turning  the  screw  a  few  turns  out  of  or 
into  it.  Into  the  upper  open  end  of  the  perpendicular  tube  fits  accurately 
a  bent  tube  g,  drawn  out  below  to  a  comparatively  small  open  point  c, 
and  having  in  its  side  a  little  above  the  point  "a  minute  needle-hole 
called  the  peephole  or  by-pass  e.  To  fix  the  apparatus  the  long 
mercury  bulb  is  placed  in  the  jacket  of  the  chamber  to  be  controlled, 
tube  a  is  connected  to  gas  supply,  tube  b  with  the  burner.  The  upper 
level  of  the  mercury  should  be  some  distance  below  the  lower  open  end 
of  tube  c.  The  burner  is  now  lit.  The  gas  passes  in  at  a  through  c 
and  e  and  out  at  b  to  the  burner.  When  the  thermometer  in  the 
interior  of  the  chamber  indicates  that  the  desired  temperature  has  been 
reached,  the  screw  d  is  turned  till  the  mercury  reaches  the  end  of  the 
tube  c.  Gas  can  only  now  pass  through  the  peephole  e,  and  the  flame 
goes  down.  The  contents  of  the  jacket  cool,  the  mercury  contracts  off 
the  end  of  tube  c,  and  the  flame  rises.  This  alternation  going  on,  the 
temperature  of  the  chamber  is  kept  very  nearly  constant.  If  the  mercury 
cuts  off  the  gas  supply  before  the  desired  temperature  is  reached,  and 
the  screw  d  is  as  far  out  as  it  will  go,  then  some  of  the  mercury  must  be 
removed.  Similarly,  if  when  the  desired  temperature  is  reached  and  the 
screw  d  is  as  far  in  as  it  can  go,  the  mercury  does  not  reach  c,  some  more 
must  be  introduced.  If  the  amount  of  gas  which  passes  through  the 


STORING  AND  INCUBATION  OF  CULTURES      87 

peephole  is  sufficient  still  to  raise  the  temperature  of  the  chamber  when 
c  is  closed  by  the  rise  of  the  mercury,  then  the  peephole  is  too  large.  Tube 
7  must  be  unshipped  and  e  plastered  over  with  sealing-wax,  which  is 
pricked,  while  still  soft,  with  a  very  line  needle.  The  gas  flame,  when 
only  the  peephole  is  supplying  gas,  ought  to  be  sufficiently  large  not  to 
be  blown  out  by  small  currents  of  air.  If  the  pressure  of  gas  supplied  to 
a  regulator  varies  much  in  the  twenty-four  hours,  a  pressure  regulator 
ought  to  be  interposed  between  the  gas  tap  and  the  instrument.  Several 
varieties  of  these  can  be  obtained.  In  all  cases  g  ought  to  be  fixed  to  b 
with  a  turn  of  wire. 

The  varieties  of  incubators  are,  as  we  have  said,  numerous. 
The    most   complicated   and   expensive   are    made   by  German 


FIG.  39. — Hearsou's  incubator  for  use  at  37°  C. 


manufacturers.  Many  of  these  are  unsatisfactory,  as  they  easily 
get  out  of  order  and  are  difficult  to  repair.  We  have  found 
those  of  Hearson  of  London  extremely  good,  and  in  proportion 
to  their  size  much  cheaper  than  the  German  articles.  They  are 
fitted  with  an  admirable  regulator.  It  is  preferable  in  using  an 
iinMibator  to  connect  the  regulator  with  the  gas  supply  and  with 
the  Bunsen  by  flexible  metal-tubing.  It  is  necessary  to  see  that 
there  is  not  too  much  evaporation  from  the  surface  of  cultures 
placed  within  incubators,  otherwise  they  may  quickly  dry  up. 
It  is  thus  advisable  to  raise  the  amount  of  water  vapour  in  the 
interior  by  having  in  the  bottom  of  the  incubator  a  flat  dish  full 
« if  water  from  which  evaporation  may  take  place.  With  tubes 


88      METHODS  OF  CULTIVATION  OF  BACTERIA 

which  will  require  to  be  long  in  the  incubator,  the  plugs  should 
be  pushed  a  little  way  into  the  tube  and  a  few  drops  of 
melted  paraffin  dropped  on  the  top  of  the  wool,  or  the  plugs 
should  be  covered  either  by  indiarubber  caps  or  by  pieces  of 
sheet  rubber  tied  over  them.  These  caps  should  be  previously 
sterilised  in  1-1000  corrosive  sublimate  and  then  dried.  Before 
they  are  placed  on  the  tube  the  cotton-wool  plug  ought  to  be 
well  singed  in  a  flame.  "  Cool  "  incubators  are  often  used  for 
incubating  gelatin  at  21°  to  22°  C.  An  incubator  of  this  kind 
fitted  with  a  low-temperature  Hearson's  regulator  is  in  the 
market. 

Method  of  Mounting  Bacterial  Cultures  as  Permanent 
Museum  Specimens  (Richard  Muir).  —  (a)  Stab  or  Stroke 
Cultures  in  Nutrient  Gelatin  or  Agar  Media. — When  the  culture 
shows  typical  characters,  further  growth  is  arrested  by  placing  the 
tube  in  a  formol  vapour  chamber,  or  by  saturating  the  cotton- 
wool plug  with  strong  formalin.  Then  leave  for  a  day  or  two. 
Make  up  the  following  : — 

(1)  Thymol  water  (saturated  in  cold)  .         .         .  100  c.c. 
Glycerin         .         .         .         .         .         .         .  20  c.c. 

Acetate  of  potash   ......  5  grms. 

Coignet's  (gold  label)  gelatin  ....  10  grms. 

Render  the  mixture  acid  to  litmus  with  acetic  acid ;  clear  with  white 
of  egg  and  filter. 

Warm  to  about  40°  C.,  and  removing  cotton-wTool  plug  from 
culture  take  a  little  of  the  preserving  fluid  in  a  pipette  and 
allow  to  run  gently  over  surface  of  medium  in  tube.  Place  in 
such  a  position  that  a  thin  layer  of  the  preserving  medium 
remains  completely  covering  the  growth  and  the  surface  of 
culture  medium.  The  gelatin  is  now  allowed  to  solidify.  Add 
three  or  four  drops  of  strong  formalin  to  the  tube,  and  fill  up  to 
within  a  quarter  of  an  inch  of  the  top  of  the  tube  with  the 
following  fluid  : — 

(2)  Thymol  water  (saturated  in  cold)    .         .         .         100  c.c. 
Glycerin         .         .         .         .         .         .         .         20  c.c. 

Acetate  of  potash    ......         5  grms. 

Cover  top  of  tube  with  a  small  piece  of  paper  so  as  to  keep  out 
dust,  allow  to  stand  for  a  day  or  two  so  that  small  air-bells  may 
rise  to  the  surface. 

To  seal  tube,  pour  melted  paraffin  gently  on  to  the  surface 
of  fluid  to  near  the  top  of  tube;  allow  to  solidify.  Cover 
paraffin  with  layer  of  alcoholic  orange  shellac  cement;  allow 


GENERAL  LABORATORY  RULES       89 

this  to  set,  and  repeat  until  the  cement  becomes  level  with  top 
of  test-tube.  When  set,  a  few  drops  of  black  lacquer  are  put  on, 
and  a  circular  cover-glass  of  about  the  same  diameter  as  the 
mouth  of  tube  is  placed  so  as  completely  to  seal  it. 

(/;)  The  following  method  is  useful  for  preparing  plate  cultures  : 
Instead  of  making  the  cultures  in  Petri's  capsules,  use  ordinary 
watch-glasses.  The  watch-glass  is  sterilised  in  a  Petri's  capsule, 
and  the  inoculated  medium  is  poured  out  into  the  watch-glass, 
allowed  to  solidify  in  the  usual  way,  and  left  in  the  Petri's 
capsule  until  the  colonies  of  growth  have  developed.  The 
watch-glass  is  now  removed  from  capsule,  and  a  layer  of  the 
preserving  gelatin  medium  (1),  to  which  have  been  added  a  few 
drops  of  strong  formalin,  is  allowed  to  spread  over  the  surface 
of  the  culture  medium.  When  the  layer  is  solidified  the  watch- 
glass  is  filled  up  with  the  same,  and  a  clean  square  or  oblong 
piece  of  glass  (which  of  course  should  be  of  slightly  larger 
diameter  than  the  watch-glass)  is  now  carefully  placed  over 
watch-glass,  care  being  taken  that  no  air-bells  are  formed.  The 
edge  of  watch-glass  should  be  closely  applied  to  the  glass  cover, 
an<l  left  in  position  until  the  gelatin  has  solidified.  The  super- 
fluous gelatin  is  now  removed,  and  the  glasses  sealed  first  with 
the  orange  shellac  cement,  then  with  black  lacquer.  It  is  now 
lii i i -lied  off  by  using  a  circular  mask  of  suitable  size. 

The  various  kinds  of  solid  media  used  in  the  cultivation  of 
bacteria,  such  as  blqod  serum,  potato,  bread  paste,  etc.,  can  be 
nvated  in  the  same  manner  with  excellent  results. 

General  Laboratory  Rules. — On  the  working  bench  of  every 
bacteriologist  there  should  be  a  large  dish  of  1-1000  solution  of 
mercuric  chloride  in  water.  Into  this  all  tubes,  vessels,  plates, 
hanging-drop  cultures,  etc.,  which  have  contained  bacteria  and 
with  which  he  has  finished,  ought  to  be  at  once  plunged  (in  the 
case  of  tubes,  the  tube  and  plug  should  be  put  in  separately). 
On  no  account  whatever  are  such  infected  articles  to  be  left 
lying  about  the  laboratory.  The  basin  is  to  be  repeatedly 
cleaned  out.  All  the  glass  is  carefully  washed  in  repeated 
changes  of  tap  water  to  remove  the  last  trace  of  perchloride  of 
mercury,  a  very  minute  quantity  of  which  is  sufficient  to  inhibit 
growth.  Old  cultures  which  have  been  stored  for  a  time,  and 
from  which  fresh  sub-cultures  have  been  made,  ought  to  be 
steamed  in  the  Koch's  steriliser  for  two  or  three  hours,  or  in  the 
autoclave  for  a  shorter  period,  and  the  tubes  thoroughly  washed 
out.  Besides  a  basin  of  mercuric  chloride  solution  for  infected 
apparatus,  etc.,  there  ought  to  be  a  second  reserved  for  the 
worker's  hands  in  case  of  any  accidental  contamination.  When, 


90      METHODS  OF  CULTIVATION  OF  BACTERIA 

as  in  public-health  work,  a  large  number  of  tubes  are  being  daily 
put  out  of  use,  they  may  be  placed  in  an  enamelled  slop-pail, 
and  this  when  full  is  placed  in  the  steam  steriliser. 

A  white  glazed  tile  on  which  a  bell-jar  can  be  set  is  very 
convenient  to  have  on  a  bench.  Infective  material  in  watch- 
glasses  can  be  placed  thus  under  cover  while  investigation  is 
going  on,  and  if  anything  is  spilled  the  whole  can  be  easily 
disinfected.  In  making  examinations  of  organs  containing 
virulent  bacteria,  the  hands  should  be  previously  dipped  in 
1-1000  mercuric  chloride  and  allowed  to  remain  wet  with  this 
solution.  No  food  ought  to  be  partaken  of  in  the  laboratory, 
and  pipes,  etc.,  are  not  to  be  laid  with  their  mouth-pieces  on 
the  bench.  No  label  is  to  be  licked  with  the  tongue.  Before 
leaving  the  laboratory  the  bacteriologist  ought  to  wash  the 
hands  and  forearms  with  1-1000  mercuric  chloride  and  then 
with  yellow  soap.  In  the  case  of  any  fluid  containing  bacteria 
being  accidentally  spilt  on  the  bench  or  floor,  1-1000  mercuric 
chloride  is  to  be  at  once  poured  on  the  spot.  The  air  of  the 
laboratory  ought  to  be  kept  as  quiet  as  possible. 


CHAPTER   III. 

i 

MICROSCOPIC  METHODS. 

The  Microscope. — For  ordinary  bacteriological  work  a  good 
microscope  is  essential.  It  ought  to  have  a  heavy  stand,  with 
rack  and  pinion  and  fine  adjustment,  a  double  mirror  (flat  on 
one  side,  concave  on  the  other),  a  good  condenser,  with  an  iris 
diaphragm,  and  a  triple  nose-piece.  It  is  best  to  have  three 
objectives,  either  Zeiss  A,  D,  and  y^-inch  oil  immersion,  or  the 
lenses  of  other  makers  corresponding  to  these.  The  oil  immer- 
sion lens  is  essential.  It  is  well  to  have  two  eye-pieces,  say 
Nos.  2  and  4  of  Zeiss  or  lenses  of  corresponding  strengths. 
The  student  must  be  thoroughly  familiar  with  the  focussing  of 
the  light  on  the  lens  by  means  of  the  condenser,  and  also  with 
the  use  of  the  immersion  lens.  It  may  here  be  remarked  that 
when  it  is  desired  to  bring  out  in  sharp  relief  the  margins  of 
unstained  objects,  e.y.  living  bacteria  in  a  fluid,  a  narrow 
aperture  of  the  diaphragm  should  be  used,  whereas,  in  the  case 
of  stained  bacteria,  when  a  pure  coloured  picture  is  desired,  the 
diaphragm  ought  to  be  widely  oj>ened.  The  flat  side  of  the 
mirror  ought  to  be  used  along  with  the  condenser.  When  the 
observer  has  finished  for  the  time  being  with  the  immersion 
lens  he  ought  to  wipe  off  the  oil  with  a  piece  of  silk  or 
very  fine  washed  linen.  If  the  oil  has  dried  on  the  lens 
it  may  be  moistened  with  xylol — never  with  alcohol,  which 
will  dissolve  the  material  by  which  the  lens  is  fixed  in  its  metal 
carrier. 

Microscopic  Examination  of  Bacteria.— 1.  Hanging-drop 
Preparations. — Micro-organisms  may  be  examined  :  (1)  alive  or 
dead  in  fluids ;  (2)  in  film  preparations ;  (3)  in  sections  of 
tissues.  In  the  two  last  cases  advantage  is  always  taken  of  the 
affinity  of  bacteria  for  certain  stains.  When  they  are  to  be 
examined  in  fluids  a  drop  of  the  liquid  may  be  placed  on  a  slide 

91 


92  MICROSCOPIC  METHODS 

and  covered  with  a  cover-glass.1  It  is  more  usual,  however,  to 
employ  hanging-drop  preparations.  The  technique  of  making 
these  has  already  been  described  (p.  69).  In  examining  them 
microscopically,  it  is  necessary  to  use  a  very  small  diaphragm. 
It  is  best  to  focus  the  edge  of  the  drop  with  a  low-power 
objective,  and,  arranging  the  slide  so  that  part  of  the  edge 
crosses  the  centre  of  the  field,  to  clamp  the  preparation  in  this 
position.  A  high-power  lens  is  then  turned  into  position,  and 
lowered  by  the  coarse  adjustment  to  a  short  distance  above  its 
focal  distance ;  it  is  now  carefully  screwed  down  by  the  fine 
adjustment,  the  eye  being  kept  at  the  tube  meanwhile.  The 
shadow  of  the  edge  will  be  first  recognised,  and  then  the  bacteria 
must  be  carefully  looked  for.  Often  a  dry  lens  is  sufficient,  but 
for  some  purposes  the  oil  immersion  is  required.  If  the  bacteria 
are  small  and  motile,  a  beginner  may  have  great  difficulty  in 
seeing  them,  and  it  is  well  to  practise  at  first  on  some  large  non- 
motile  form,  such  as  anthrax.  In  fluid  preparations  the  natural 
appearance  of  bacteria  may  be  studied,  and  their  rate  of  growth 
determined.  The  great  use  of  such  preparations,  however,  is  to 
find  whether  or  not  the  bacteria  are  motile,  and  for  determining 
this  point  it  is  advisable  to  use  either  broth  or  agar  cultures  not 
more  than  twenty-four  hours  old.  In  the  latter  case  a  small 
fragment  of  growth  is  broken  down  in  broth  or  in  sterile  wrater. 
Sometimes  it  is  an  advantage  to  colour  the  solution  in  which 
the  hanging-drop  is  made  up  with  a  minute  quantity  of  an 
aniline  dye,  say  a  small  crystal  of  gentian  violet  to  100  c.c.  of 
bouillon.  Such  a  degree  of  dilution  wdll  not  have  any  effect  on 
the  vitality  of  the  bacteria.  Ordinarily,  living  bacteria  will  not 
take  up  a  stain,  but  even  though  they  do  not,  the  contrast 
between  the  unstained  bacteria  and  the  tinted  fluid  will  enable 
the  observer  more  easily  to  recognise  them.  In  determining 
whether  or  not  a  bacterium  is  motile,  great  difficulty  is  often 
experienced  in  distinguishing  between  true  motion  and  Brownian 
movement,  especially  if  the  organism  be  small.  The  essential 
criterion  to  be  fulfilled  is  that  the  bacteria  shall  be  moving  in 
all  directions,  the  observation  of  individuals  lying  close  together 
starting  to  move  in  opposite  directions  being  important.  The 
observation  of  hanging-drop  preparations  must  be  correlated 
with  the  results  of  staining  for  the  presence  of  flagella  which,  so 
far  as  is  known,  are  present  in  all  motile  forms. 

Within  recent  years    the    method  of  observing  living  micro- 

1  In  bacteriological  work  it  is  essential  that  cover-glasses  of  No.  1  thickness 
(i.e.  '14  mm.  thick)  should  be  used,  as  those  of  greater  thickness  are  not 
suitable  for  a  jViuch  lens. 


FILM  PREPARATIONS  93 

organism*  by  oblique  illumination  has  been  much  practised,  and 
a  number  of  substage  condensers  are  in  the  market,  by  means  of 
which  this  is  effected.  The  general  principle  involved  in  these 
instruments  is  to  stop  out  the  rays  passing  directly  towards  the 
tube  of  the  microscope,  and  to  arrange  for  light  being  thrown 
obliquely  on  bacteria  mounted  in  a  drop  of  fluid  between  a  slide 
and  cover-glass.  The  bacteria  disperse  these  rays  in  all  direc- 
tions, and  some  passing  up  through  the  lens  are  focussed  by  it* 
The  organisms  thus  appear  as  brightly  illumined  objects  on  a 
dark  background.  The  method  has  been  employed  for  bacteria 
in  general,  and  especially  for  the  demonstration  of  the  spirochcbte 
pallida  in  secretions  as  a  means  of  diagnosis.  Generally  speak- 
ing, the  internal  structure  of  the  organisms  under  observation  is 
well  brought  out. 

2.  Film  Preparations.— (a)  Dry  Method. — This  is  the  most 
extensively  applicable  method  of  microscopically  examining 
bacteria.  Fluids  containing  bacteria,  such  as  blood,  pus, 
scrapings  of  organs,  can  be  thus  investigated,  as  also  cultures 
in  fluid  and  solid  media.  The  first  requisite  is  a  perfectly  clean 
cover-glass.  Many  methods  are  recommended  for  obtaining 
.such.  The  test  of  this  being  accomplished  is  that,  when  the 
drop  of  fluid  containing  the  bacteria  is  placed  upon  the  glass,  it 
can  be  uniformly  spread  with  the  platinum  needle  all  over  the 
surface  without  showing  any  tendency  to  retract  into  droplets. 
The  best  method  is  that  recommended  by  Van  Ermengem.  The 
cover-glasses  are  placed  for  some  time  in  a  mixture  of  con- 
centrated sulphuric  acid  6  parts,  potassium  bichromate  6  parts, 
water  100  parts,  then  washed  thoroughly  in  water  and  stored  in 
absolute  alcohol.  For  use,  a  cover-glass  is  either  dried  by 
wiping  with  a  clean  duster  or  is  simply  allowed  to  dry.  This 
method  will  amply  repay  the  trouble,  and  really  saves  time  in 
the  end.  A  clean  cover  having  been  obtained,  the  film  pre- 
paration can  now  be  made.  If  a  fluid  is  to  be  examined  a 
loopful  may  be  placed  on  the  cover-glass,  and  either  spread 
out  over  the  surface  with  the  needle,  or  another  clean  cover 
may  be  placed  on  the  top  of  the  first,  the  drop  thus  spread 
out  between  them  and  the  two  then  drawn  apart.  When 
a  culture  on  a  solid  medium  is  to  be  examined,  a  loopful  of 
distilled  water  is  placed  on  the  cover-glass,  and  a  minute  particle 
of  growth  rubbed  up  in  it  and  spread  over  the  glass.  The  great 
mistake  made  by  beginners  is  to  take  too  much  of  the  growth. 
The  point  of  the  straight  needle  should  just  touch  the  surface 
<>t'  the  culture,  and  when  this  is  rubbed  up  in  the  droplet  of 
water  and  the  film  dried,  there  should  be  an  opaque  cloud  just 


94  MICROSCOPIC  METHODS 

visible  on  the  cover-glass.  When  the  film  has  been  spread,  it 
must  next  be  dried  by  being  waved  backwards  and  forwards  at 
arm's-length  above  a  Bunsen  flame.  The  film  must  then  be  fixed 
on  the  glass  by  being  passed  three  or  four  times  slowly  through 
the  flame.  In  doing  this  a  good  plan  is  to  hold  the  cover-glass 

between  the  right  forefinger 
and  thumb  ;  if  the  fingers  just 
escape  being  burned  no  harm 
will  accrue  to  the  bacteria  in 

.    FIG.  40. — Cornet's  forceps  for  holding  ',.         p,  r         ,-L-  i 

cover-glasses.  ^n  making  films  01  a  thick 

fluid  such  as  pus,  it  is  best  to 

spread  it  out  on  one  cover  with  the  needle.  The  result  will  be 
a  film  of  irregular  thickness,  but  sufficiently  thin  at  many  parts 
for  proper  examination.  Scrapings  of  organs  may  be  smeared 
directly  on  the  cover-glasses. 

In  the  case  of  blood,  a  fairly  large  drop  should  be  allowed  to 
spread  itself  between  two  clean  cover-glasses,  which  are  then  to 
be  slipped  apart,  and  being  held  between  the  forefinger  and 
thumb  are  to  be  dried  by  a  rapid  to-and-fro  movement  in  the 
air.  A  film  prepared  in  this  way  may  be  too  thick  at  one  edge, 
but  at  the  other  is  beautifully  thin.  If  it  is  desired  to  preserve 
the  red  blood  corpuscles  in  such  a  film  it  may  be  fixed  by  one 
of  the  following  methods :  by  being  placed  (a)  in  a  hot-air 
chamber  at  1 20°  C.  for  half  an  hour ;  (b)  in  a  mixture  of  equal 
parts  of  alcohol  and  ether  for  half  an  hour,  then  washed  and 
dried ;  (c)  in  formol-alcohol  (Gulland)  (formalin  1  part,  absolute 
alcohol  9  parts)  for  five  minutes,  then  washed  and  dried  ;  or  (d) 
in  a  saturated  solution  of  corrosive  sublimate  for  two  or  three 
minutes,  then  washed  well  in  running  water  and  dried.  (Fig.  69 
shows  a  film  prepared  by  the  last  method.)  In  using  the 
Romanowsky  stains  no  previous  fixation  is  necessary  (vide  infra). 
In  the  case  of  urine,  the  specimen  must  be  allowed  to  stand,  and 
films  made  from  any  deposit  which  occurs ;  or,  what  is  still 
better,  the  urine  is  centrifugalised,  and  films  made  from  the 
deposit  which  forms.  After  dried  films  are  thus  made  from 
urine  it  is  an  advantage  to  place  a  drop  of  distilled  water  on  the 
film  and  heat  gently  to  dissolve  the  deposit  of  salts ;  then  w^ash 
in  water  and  dry.  In  this  way  a  much  clearer  picture  is 
obtained  when  the  preparation  is  stained. 

Within  recent  years  it  has  become  common  to  make  blood 
films  on  ordinary  microscopic  slides  instead  of  upon  cover- 
glasses.  Here  the  slides  must  be  clean.  This  can  be  effected  by 
washing  thoroughly  first  with  weak  alkali  and  then  with  water 


FILM  PREPAKATIONS  95 

and  storing  in  alcohol.  For  use,  a  slide  is  taken  from  the 
alcohol  and  the  fluid  adhering  to  it  set  on  fire  and  allowed  to 
burn  off,  a  dry  clean  slide  being  thus  obtained.  To  make  a  film 
on  such,  a  small  drop  of  blood  is  placed  near  one  end,  the  edge 
of  a  second  clean  slide  is  lowered  through  the  drop  on  to  the 
surface  of  the  glass  on  which  the  blood  has  been  placed.  This 
second  slide  is  held  at  an  angle  to  the  first,  and  the  droplet  of 
blood  by  capillarity  spreads  itself  in  the  angle  between  the  two 
slides.  The  edge  of  the  second  slide  is  then  stroked  along  the 
surface  of  the  first  slide,  and  the  blood  is  spread  out  in  a  film 
whose  thickness  can  be  regulated  by  the  angle  formed  by  the 
second  slide.  Large-sized  films  can  thus  be  obtained,  and  when 
these  are  stained  they  are  often  examined  without  any  cover- 
glass  being  placed  upon  them.  A  drop  of  cedar  oil  is  placed  on 
the  preparation,  and  after  use  this  can  be  removed  by  the  careful 
application  of  xylol. 

Films  dried  and  fixed  by  the  above  methods  are  now  ready  to 
be  stained  by  the  methods  to  be  described  below. 

(/;)  Wet-  M'tlioil. — If  it  is  desired  to  examine  the  fine 
histological  structure  of  the  cells  of  a  discharge  as  well  as  to 
investigate  the  bacteria  present,  it  is  advisable  to  substitute 
"  wet "  films  for  the  "  dried  "  films,  the  preparation  of  which  has 
been  described.  The  nuclear  structure,  mitotic  figures,  etc.,  are 
by  this  method  well  preserved,  whereas  these  are  considerably 
distorted  in  dried  films.  The  initial  stages  in  the  preparation 
of  wet  films  are  the  same  as  above,  but  instead  of  being  dried 
in  air  they  are  placed,  while  still  wet,  film  downwards  in 
the  fixative.  The  following  are  some  of  the  best  fixing 
methods : — 

(a)  A  saturated  solution  of  perohloride  of  mercury  in  '75  per  cent, 
sodium  chloride  ;  fix  for  live  minutes.     Then  place  the  films  for  half  an 
hour,  with  occasional  gentle  shaking,  in  '75  per  cent,  sodium  chloride 
solution  to  wash  out  the  corrosive  sublimate  ;  they  are  thereafter  washed 
in  successive  strengths  of  methylated  spirit.     After  this  treatment  the 
films  are  stained  and  treated  as  it'  they  were  sections. 

(b)  Formol-alcohol — formalin  1   part,  absolute  alcohol   9.     Fix   films 
for  three  minutes ;   then  wash  well  in  methylated   spirit.     This  is  an 
excellent  and  very  rapid  method. 

(c)  Another  excellent  method  of  fixing  has  been  devised  by  Gulland. 
The  fixing  solution  has  the  composition — absolute  alcohol  25  c.c.,  pure 
ether  25  c.c.,  alcoholic  solution  of  corrosive  sublimate  (2  grins,  in  10  c.c. 
o(  alcohol)' about  5  drops.     The  films  are  placed  in  this  solution  for  five 
minutes  or  longer.     They  are  then  washed  well  in  water,  and  are  ready 
for  staining.     A  contrast  stain  can  be  applied  at  the  same  time  as  the 
fixing   solution,  by  saturating  the  25  c.c.  of  alcohol  with  eosin  before 
mixing.     Thereafter  the  bacteria,  etc.,  may  be  stained  with  methylene- 
blue  or  otlu-i  >t;dn,  as  described  below.     This  method  has  the  advantage 


96  MICROSCOPIC  METHODS 

over  (a)  that,  as  a  small  amount  of  corrosive  sublimate  is  used,  less 
washing  is  necessary  to  remove  it  from  the  preparation,  and  deposits  are 
less  liable  to  occur. 

3.  Examination  of  Bacteria  in  Tissues. — For  the  examina- 
tion of  bacteria  in  the  tissues,  the  latter  must  be  fixed  and 
hardened,  in  preparation  for  being  cut  with  a  microtome. 
Fixation  consists  in  so  treating  a  tissue  that  it  shall  permanently 
maintain,  as  far  as  possible,  the  condition  it  was  in  when  re- 
moved from  the  body.  Hardening  consists  in  giving  such  a 
fixed  tissue  sufficient  consistence  to  enable  a  thin  section  of  it 
to  be  cut.  A  tissue,  after  being  hardened,  may  be  cut  in  a 
freezing  microtome  (e.g.  Cathcart's  or  one  of  the  newer  instru- 
ments in  which  the  freezing  is  accomplished  by  compressed 
carbonic  acid  gas),  but  far  finer  results  can  be  obtained  by 
embedding  the  tissue  in  solid  paraffin  and  cutting  with  some  of 
the  more  delicate  microtomes  of  which,  for  pathological  purposes, 
the  small  Cambridge  rocker  is  by  far  the  best.  For  bacterio- 
logical purposes  embedding  in  celloidin  is  not  advisable,  as  the 
celloidin  takes  on  the  aniline  dyes  which  are  used  for  staining 
bacteria,  and  is  apt  thus  to  spoil  the  preparation,  and  besides, 
thinner  sections  can  be  obtained  by  the  paraffin  method. 

The  Fixation  and  Hardening  of  Tissues. — The  following  are 
amongst  the  best  methods  for  bacteriological  purposes  : — 

(a)  Absolute  alcohol  may  be  used  for  the  double  purpose  of  fixing  and 
hardening.     If  the  piece  of  tissue  is  not  more  than  £  inch  in  thickness,  it 
is  sufficient  to  keep  it  in  this  reagent  for  a   few  hours.     If  the   pieces 
are  thicker  a  longer  exposure  is  necessary,  and  in  such  cases  it  is  better 
to  change  the  alcohol  at  the  end  of  the  first  twenty-four  hours.     The 
tissue  must  be  tough  without  being  hard,  and  the  necessary  consistence, 
as  estimated  by  feeling  with  the  fingers,  can    only  be  judged   of  after 
some  experience.     If  the  tissues  are  not  to  be  cut  at  once,  they  may  be 
preserved  in  50  per  cent,  spirit. 

(b)  Formol-alcohol — formalin  1,  absolute  alcohol  9.     Fix  for  not  more 
than  twenty- four  hours  ;  then  place  in  absolute  alcohol  if  the  tissue  is 
to  be  embedded  at  once,  in  50  per  cent,  spirit  if  it  is  to  be  kept  for  some 
time.     For  small  pieces  of  tissue  fixation  for  twelve  hours  or  even  less  is 
sufficient.     The  method  is  a  rapid  and  very  satisfactory  one. 

(c)  Corrosive  sublimate  is  an  excellent  fixing  agent.     It  is  best   used 
as  a  saturated  solution  in   '75  per  cent,  sodium  chloride  solution.     Dis- 
solve  the  sublimate   in   the   salt   solution   by   heat ;   the   separation   of 
crystals  on  cooling   shows   that   the   solution   is   saturated.     For  small 
pieces  of  tissue  |  inch  in  thickness,  twelve  hours'  immersion  is  'sufficient. 
If  the  pieces  are  larger,  twenty-four  hours  is   necessary.     They  should 
then  be  tied  up  in  a  piece  of  gauze,  and  placed  in  a  stream  of  running 
water  for  from  twelve  to  twenty-four  hours,  according  to  the  size  of  the 
pieces,  to  wash  out  the  excess  of  sublimate.     They  are  then  placed  for 
twenty-four  hours   in   each   of   the   following   strengths   of  methylated 


THE  CUTTING  OF  SECTIONS  97 

spirit  (free  from  naphtha1)  :  30  per  cent.,  60  per  cent.,  and  90  per  cent. 
Finally  they  are  placed  in  absolute  alcohol  for  twenty- four  hours  and 
are  then  ready  to  be  prepared  for  cutting. 

If  the  tissue  is  very  small,  as  in  the  case  of  minute  pieces  removed 
for  diagnosis,  the  stages  may  be  all  compressed  into  twenty-four  hours. 
In  fact,  after  fixation  in  corrosive  the  tissue  may  be  transferred  directly 
to  absolute  alcohol,  the  perchloride  of  mercury  being  removed  after  the 
sections  are  cut,  as  will  be  afterwards  described. 

(d)  Methylated  spirit.—  Small  pieces  of  tissue  may  be  placed  in 
methylated  spirit,  which  is  to  be  changed  after  the  first  day.  In  from 
six  to  seven  days  they  will  be  hardened.  If  the  pieces  are  large,  a 
longer  time  is  necessary. 

The  Cutting  of  Sections. — 1.  By  Means  of  the  Freezing 
Microtome. — Pieces  of  tissue  hardened  by  any  of  the  above 
methods  must  have  all  the  alcohol  removed  from  them  by  wash- 
ing in  running  water  for  twenty-four  hours.  They  are  then 
placed  for  from  twelve  to  twenty-four  hours  (according  to  their 
size)  in  a  thick  syrupy  solution  containing  two  parts  of  gum 
arabic  and  one  part  of  sugar.  They  are  then  cut  on  a  freezing 
microtome  and  placed  for  a  few  hours  in  a  bowl  of  water  so  that 
the  gum  and  syrup  may  dissolve  out.  They  are  then  stained,  or 
they  may  be  stored  in  methylated  spirit. 

2.  Embedding  and  Cutting  in  Solid  Paraffin. — This  method 
gives  by  far  the  finest  results,  and  should  always  be  adopted 
when  practicable.  The  principle  is  the  impregnation  of  the 
tissue  with  paraffin  in  the  melted  state.  This  paraffin  when  it 
solidifies  gives  support  to  all  the  -tissue  elements.  The  method 
involves  that,  after  hardening,  the  tissue  shall  be  thoroughly 
dehydrated,  and  then  thoroughly  permeated  by  some  solvent 
of  paraffin  which  will  expel  the  dehydrating  fluid  and  prepare 
for  the  entrance  of  the  paraffin.  The  solvents  most  in  use  are 
chloroform,  cedar  oil,  xylol,  and  turpentine ;  of  these,  chloroform 
and  cedar  oil  are  the  best,  the  former  being  preferred,  as  it  per- 
meates the  tissue  more  rapidly.  The  more  gradually  the  tissues 
are  changed  from  reagent  to  reagent  in  the  processes  to  be  gone 
through,  the  more  successful  is  the  result.  A  necessity  of  the 
process  is  an  oven  with  hot- water  jacket,  in  which  the  paraffin 
can  be  kept  at  a  constant  temperature  just  above  its  melting- 
point,  a  gas  regulator,  e.g.  Reichert's,  being  of  course  necessary. 
The  tissues  occurring  in  pathological  work  have  a  tendency  to 

1  In  Britain  ordinary  commercial  methylated  spirit  has  mineral  naphtha  added 
to  it  to  discourage  its  being  used  as  a  beverage.  The  naphtha  being  insoluble 
in  water  a  milky  fluid  results  from  the  dilution  of  the  spirit.  By  law,  chemists 
can  only  sell  8  ounces  of  pure  spirit  at  a  time.  Most  pathological  laboratories 
are,  however,  permitted  by  the  Excise  to  buy  "industrial  spirit,"  which 
contains  only  one-nineteenth  of  naphtha. 


98  MICROSCOPIC  METHODS 

become  brittle  if  overheated,  and  therefore  the  best  results  are 
obtained  by  using  paraffin  melting  at  a  somewhat  low  tempera- 
ture. We  have  used  for  some  years  a  mixture  of  one  part  of 
paraffin,  melting  at  48°,  and  two  parts  of  paraffin  melting  at 
54°  C.  This  mixture  has  a  melting-point  between  52°  and 
53°  C.,  and  it  serves  all  ordinary  purposes  well.  An  excellent 
quality  of  paraffin  is  that  known  as  the  "  Cambridge  paraffin," 
but  many  scientific-instrument  makers  supply  paraffins  which,  for 
ordinary  purposes,  are  quite  as  good,  and  much  cheaper.  The 
successive  steps  in  the  process  of  paraffin  embedding  are  as 
follows  :  l — 

1.  Pieces  of  tissue,   however  hardened,   are  placed  in  fresh  absolute 
alcohol  for  twenty-four  hours  in  order  to  their  complete  dehydration. 

2.  Transfer  now  to  a  mixture  of  equal  parts  of  absolute  alcohol  and 
chloroform  for  twenty- four  hours. 

3.  Transfer  to  pure  chloroform  for  twenty-four  hours  or  longer.     At 
the  end  of  this  time  the  tissues  should  sink  or  float  heavily. 

4.  Transfer  now  to  a  mixture  of  equal  parts  of  chloroform  and  paraffin 
and  place  on  the  top  of  the  oven  for  from  twelve  to  twenty-four  hours. 
If  the  temperature  there  is  not  sufficient  to  keep  the  mixture  melted 
then  they  must  be  put  inside. 

5.  Place  in  pure  melted  paraffin  in  the  oven  for  twenty-four  hours. 
For  holding  the  paraffin  containing  the  tissues,  small  tin  dishes  such  as  are 
used  by  pastry-cooks  will   be   found  very  suitable.     There  must   be   a 
considerable  excess  of  paraffin  over  the  bulk  of  tissue  present,  otherwise 
sufficient  chloroform  will  be  present  to  vitiate  the  final  result  and  not 
give  the  perfectly  hard  block  obtained  with  pure  paraffin.     With  ex- 
perience,  the  persistence  of  the  . slightest  trace   of  chloroform   can  be 
recognised  by  smell. 

In  the  case  of  very  small  pieces  of  tissue  the  time  given  for  each  stage 
may  be  much  shortened,  and  where  haste  is  desirable  Nos.  2  and  4  may 
be  omitted.  Otherwise  it  is  better  to  carry  out  the  process  as  described. 

6.  Cast  the  tissues  in  blocks  of  paraffin  as  follows  :  Pairs  of  L-shaped 
pieces  of  metal  made  for  the  purpose  by  instrument  makers  must  be  at 
hand.     By  laying  two  of  these  together  on  a  glass  plate,  a  rectangular 
trough  is  formed.     This  is  filled  with  melted  paraffin  taken  from  a  stock 
in  a  separate  dish.     In  it  is  immersed  the  piece  of  tissue,  which  is  lifted 
out   of  its   pure   paraffin  bath  with   heated  forceps.     The  direction  in 
which  it  is  to  be  cut  must  be  noted  before  the  paraffin  becomes  opaque. 
When  the  paraffin  has  begun  to  set,   the  'glass  plate  and  trough  have 
cold  water  run  over  them.     When  the  block  is  cold,  the  metal  L's  are 
broken  off,  and,  its  edges  having  been  pared,  it  is  stored  in  a  pill-box. 

The  Cutting  of  Paraffin  Sections. — Sections  must  be  cut  as 

1  While  the  method  given  is  sufficient  for  ordinary  purposes,  a  more  elaborate 
technique  is  necessary  if  it  is  desired  that  no  changes  shall  take  place  in  the 
tissue.  Thus  after  fixation  the  tissue  must  be  taken  up  to  absolute  alcohol 
through  successive  dilutions  of  spirit,  but  differing  from  each  other  by  more 
than  10  per  cent.  Again,  when  alcohol  has  been  replaced  by  chloroform  the 
latter  must  be  saturated  with  chips  of  paraffin,  first  at  even  temperature,  then 
at  37°  C.,  and  must  be  kept  at  55°  C.  as  short  a  time  as  possible. 


THE  CUTTING  OF  SECTIONS  99 

thin  as  possible,  the  Cambridge  rocking  microtome  being,  on 
the  whole,  most  suitable.  They  should  not  exceed  8  /*  in  thick- 
ness, and  ought,  if  possible,  to  be  about  4  /x.  For  their  mani- 
pulation it  is  best  to  have  two  needles  on  handles,  two  camel's- 
liair  brushes  on  handles,  and  a  needle  with  a  rectangle  of  stiff 


Fin.  41. — Needle  with  square  of  paper  on  end  for  manipulating 
paraffin  sections. 

writing  paper  fixed  on  it  as  in  the  diagram  (Fig.  41).  When 
cut,  sections  are  floated  on  the  surface  of  a  beaker  of  water  kept 
at  a  temperature  about  10°  C.  below  the  melting-point  of  the 
paraffin.  On  the  surface  of  the  warm  water  they  become 
perfectly  flat. 

Fixation  on  Ordinary  Slides,  (a)  Gulland's  Method.—  A  supply  of 
slides  well  cleaned  being  at  hand,  one  of  them  is  thrust  obliquely  into 
the  water  below  the  section,  a  corner  of  the  section  is  fixed  on  it  with  a 
needle  and  the  slide  withdrawn.  The  surplus  of  water  being  wiped  off 
witli  a  cloth,  the  slide  is  placed  on  a  support,  with  the  section  down- 
wauls,  and  allowed  to  remain  on  the  top  of  the  paraffin  oven  or  in  a 
bacteriological  incubator  for  from  twelve  to  twenty- four  hours.  It  will 
then  be  sufficiently  fixed  on  the  slide  to  withstand  all  the  manipulations 
necessary  during  staining  and  mounting. 

(V)  Fixation  by  Mann's  Method. — This  has  the  advantage  of  being 
more  rapid  than  the  previous  one.  A  solution  of  albumin  is  prepared 
by  mixing  the  white  of  a  fresh  egg  with  ten  parts  of  distilled  water  and 
filtering.  Slides  are  made  perfectly  clean  with  alcohol.  One  is  dipped 
into  the  solution  and  its  edge  is  then  drawn  over  one  surface  of  another 
slide  so  as  to  leave  on  it  a  thin  film  of  albumin.  This  is  repeated  with 
the  others.  As  each  is  thus  coated  it  is  leant,  with  the  film  down- 
wards, on  a  ledge  till  dry,  and  then  the  slides  are  stored  in  a  wide 
stoppered  jar  till  needea.  The  floating  out  is  performed  as  before. 
The  albuminised  side  of  the  slide  is  easily  recognised  by  the  fact  that 
if  it  is  breathed  on,  the  breath  does  not  condense  on  it.  The  great 
advantage  of  this  method  is  that  the  section  is  fixed  after  twenty  to 
thirty  minutes'  drying  at  37°  C.  If  the  tissue  has  been  hardened  in  any 
of  the  bichromate  solutions  and  embedded  in  paraffin,  this  or  some 
corresponding  method  of  fixing  the  sections  on  the  slide  must  be  used. 

Preparation  of  Paraffin  Sections  for  Staining. — Before  stain- 
ing, the  paraffin  must  be  removed  from  the  section.  This  is 
best  done  by  dropping  on  xylol  out  of  a  drop  bottle.  When  the 
paraffin  is  dissolved  out,  the  superfluous  xylol  is  wiped  off  with 
a  cloth  and  a  little  absolute  alcohol  dropped  on.  When  the 
xylol  is  removed,  the  superfluous  alcohol  is  wiped  off  and  a 
little  50  per  cent,  methylated  spirit  dropped  on.  During  these 


100  MICROSCOPIC  METHODS 

procedures  sections  must  on  no  account  be  allowed  to  dry. 
The  sections  are  now  ready  to  be  stained.  Deposits  of  crystals 
of  corrosive  sublimate  often  occur  in  sections  which  have  been 
fixed  by  this  reagent.  These  can  be  removed  by  placing  the 
sections,  before  staining,  for  a  few  minutes  in  equal  parts  of 
Gram's  iodine  solution  (p.  106)  and  water,  and  then  washing  out 
the  iodine  with  methylated  spirit. 

To  save  repetition,  we  shall  in  treating  of  stains  suppose  that, 
with  paraffin  sections,  the  above  preliminary  steps  have  already 
been  taken,  and  further,  that  sections  cut  by  a  freezing  microtome 
are  also  in  spirit  and  water. 

Dehydration  and  Clearing. — It  is  convenient,  first  of  all,  to 
indicate  the  final  steps  to  be  taken  after  a  specimen  is  stained. 
Dry  films  after  being  stained  are  washed  in  water,  dried  and 
mounted  in  xylol  balsam;  wet  films  and  sections  must  be 
dehydrated,  cleared,  and  then  mounted  in  xylol  balsam. 

Dehydration  is  most  commonly  effected  with  absolute  alcohol. 
Alcohol,  however,  sometimes  decolorises  the  stained  organisms 
more  than  is  desirable,  and  therefore  Weigert  devised  the 
following  method  of  dehydrating  and  clearing  by  aniline  oil. 
which,  though  it  may  decolorise  somewhat,  does  not  do  so  to  the 
same  extent  as  alcohol.  As  much  as  possible  of  the  water  being 
removed,  the  section  placed  on  a  slide  is  partially  dried  by 
draining  with  fine  blotting-paper.  Some  aniline  oil  is  placed  on 
the  section  and  the  slide  moved  to  and  fro.  The  section  is 
dehydrated  and  becomes  clear.  The  process  may  be  accelerated 
by  heating  gently.  The  preparation  is  then  treated  with  a 
mixture  of  two  parts  of  aniline  oil  and  one  part  of  xylol,  and 
then  with  xylol  alone,  after  which  it  is  mounted  in  xylol  balsam. 
Balsam  as  ordinarily  supplied  has  often  an  acid  reaction,  and 
preparations  stained  with  aniline  dyes  are  apt  to  fade  when 
mounted  in  it.  It  is  accordingly  a  great  advantage  to  use  the 
acid-free  balsam  supplied  by  Griibler.  Paraffin  sections  can 
usually  be  dehydrated  and  cleared  by  the  mixture  of  aniline  oil 
and  xylol  alone. 

Sections  stained  for  bacteria  should  always  be  cleared,  at 
least  finally,  in  xylol,  as  it  dissolves  out  aniline  dyes  less  readily 
than  such  clearing  reagents  as  clove  oil,  etc.  Xylol,  however, 
requires  the  previous  dehydration  to  have  been  more  complete 
than  clove  oil,  which  will  clear  a  section  readily  when  the 
dehydration  has  been  only  partially  effected  by,  say,  methylated 
spirit.  If  a  little  decolorisation  of  a  section  is  still  required 
before  mounting,  clove  oil  may  be  used  to  commence  the 
clearing,  the  process  being  finished  with  xylol.  With  a  little 


THE  STAINING  OF  BACTERIA  101 

experience  the  process  of   decolorisatioii   can  be  judged  of   •>/ 
observing  the  appearances  under  a  low  objective. 

THE  STAINING  OF  BACTERIA. 

Staining  Principles. — To  speak  generally,  the  protoplasm  of 
bacteria  reacts  to  stains  in  a  manner  similar  to  the  nuclear 
cliiomatin,  though  sometimes  more  and  sometimes  less  actively. 
The  bacterial  stains  par  excellence  are  the  basic  aniline  dyes. 
These  dyes  are  more  or  less  complicated  compounds  derived 
from  the  coal-tar  product  aniline  (C6H5 .  NH2).  Many  of  them 
have  the  constitution  of  salts.  Such  compounds  are  divided 
into  two  groups  according  as  the  staining  action  depends  on  the 
basic  or  the  acid  portion  of  the  molecule.  Thus  the  acetate  of 
rosaniline  derives  its  staining  action  from  the  rosaniline.  It 
is  therefore  called  a  basic  aniline  dye.  On  the  other  hand, 
ammonium  picrate  owes  its  action  to  the  picric  acid  part  of  the 
molecule.  It  is  therefore  termed  an  acid  aniline  dye.  These 
t  \\ « »  groups  have  affinities  for  different  parts  of  the  .animal  cell. 
The  basic  stains  have  a  special  affinity  for  the  nuclear  chromatin, 
the  acid  for  the  protoplasm  and  various  formed  elements.  Thus 
it  is  that  the  former — the  basic  aniline  dyes — are  especially  the 
bacterial  stains. 

The  number  of  basic  aniline  stains  is  very  large.  The  following  are 
the  most  commonly  used  : — 

i  Stains. — Methyl- violet,  R-5R  (synonyms:    Hoffmann's  violet, 
dahlia). 

'  ;<'iitian-violet  (synonyms  :  benzyl-violet,  Pyoktanin). 
Crystal  violet. 

Blue  Stains.  — Methylene-blue T  (synonym  :  phenylene-blue). 
Victoria-blue. 
Thionin-blue. 
Red  Stains.—  Basic  fuchsin  (synonyms  :  basic  rubin,  magenta). 

Safraiiin  (synonyms  :  fuchsia,  Girofle). 

Broivn    Stain.  —  Bismarck  -  brown    (synonyms  :    vesuvin,    phenylene- 
brown). 

It  is  of  the  greatest  importance  that  the  stains  used  by  the 
bacteriologist  should  be  good,  and  therefore  it  is  advisable  to 
obtain  those  prepared  by  Griibler  of  Leipzig. 

Of  the  stains  specified,  the  violets  and  reds  are  the  most 
intense  in  action,  especially  the  former.  It  is  thus  easy  in  using 
tin-in  to  overstain  a  specimen.  Of  the  blues,  methylene-blue 
probably  gives  the  best  differentiation  of  structure,  and  it  is 

1  This  is  to  lie  distiiiguishc'd  from  methyl-blue,  which  is  a  different  com- 
pound. 


102 


MICROSCOPIC  METHODS 


difficult  to  overstain  with  it.  Thionin-blue  also  gives  good  dif- 
ferentiation and  does  not  readily  overstain.  Its  tone  is  deeper 
than  that  of  methylene-blue,  and  it  approaches  the  violets  in  tint. 
Bismarck-brown  is  a  weak  stain,  but  is  useful  for  some  purposes. 
Formerly  it  was  much  used  in  photomicrographic  work,  as  it  was 
less  actinic  than  the  other  stains.  It  is  not,  however,  needed 
now,  on  account  of  the  improved  sensitiveness  of  plates. 

It  is  most  convenient  to  keep  saturated  alcoholic  solutions 
of  the  stains  made  up,  and  for  use  to  filter  a  little  into  about 
ten  times  its  bulk  of  distilled  water  in  a  watch-glass.  A  solution 
of  good  body  is  thus  obtained.  Most 
bacteria  (except  those  of  tubercle, 
leprosy,  and  a  few  others)  will  stain  in 
a  short  time  in  such  a  fluid.  Watery 
solutions  may  also  be  made  up,  e.g.  a 
saturated  watery  solution  of  methylene- 
blue  or  a  1  per  cent,  solution  of 
gentian-violet.  Stains  must  always  be 
filtered  before  use ;  otherwise  there 
may  be  deposited  on  the  preparation 
granules  which  it  is  impossible  to  wash 
off.  The  violet  stains  in  solution  in 
water  have  a  great  tendency  to  decom- 
pose. Only  small  quantities  should 
therefore  be  prepared  at  a  time. 

The  Staining  of  Cover-glass  Films. 
— Films  are  made  from  cultures  as 
described  above.  The  cover-glass  may 
be  floated  on  the  surface  of  the  stain 
in  a  watch-glass,  or  the  cover-glass  held 
in  Cornet's  forceps  with  film  side 
uppermost  may  have  as  much  stain 
poured  on  it  as  it  will  hold.  When 
the  preparation  has  been  exposed  for 
the  requisite  time,  usually  a  few 
minutes,  it  is  well  washed  in  tap  water 

in  a  bowl,  or  with  distilled  water  with  such  a  simple  siphon 
arrangement  as  that  figured  (Fig.  42).  The  figure  explains  itself. 
When  the  film  has  been  washed  the  surplus  of  water  is  drawn  off 
with  a  piece  of  filter-paper,  the  preparation  is  carefully  dried 
high  over  a  flame,  a  drop  of  xylol  balsam  is  applied,  and  the 
cover-glass  mounted  on  a  slide.  It  is  sometimes  advantageous 
to  examine  films  in  a  drop  of  water  in  place  of  balsam.  The 
films  can  be  subsequently  dried  and  mounted  permanently. 


FIG.  42.  —  Siphon  wash- 
bottle  for  distilled  water 
used  in  washing  prepara- 
tions. 


MORDANTS  AND  DECOLORISING  AGENTS     103 

Films  of  fluids  from  the  body  (blood,  pus,  etc.)  can  be 
U'Mierally  stained  in  the  same  way,  and  this  is  often  quite 
sufficient  for  diagnostic  purposes.  The  blue  dyes  are  here 
preferable,  as  they  do  not  readily  overstain.  In  the  case  of  such 
fluids,  if  the  histological  elements  also  claim  attention  it  is  best 
first  to  stain  the  cellular  protoplasm  with  1-2  per  cent,  watery 
solution  of  eosiu  (which  is  an  acid  dye),  and  then  to  use  a  blue 
which  will  stain  the  bacteria  and  the  nuclei  of  the  cells.  The 
Romano wsky  stains  (vide  p.  113)  are  here  most  useful,  as  by  these 
the  preparations  are  fixed  as  well  as  stained.  Fixation  by  heat, 
which  is  apt  to  injure  delicate  cellular  structures,  is  thus  avoided. 
In  the  case  of  films  made  from  urine,  where  there  is  little  or 
no  albuminous  matter  present,  the  bacteria  may  be  imperfectly 
fixed  on  the  slide,  and  are  thus  apt  to  be  washed  off.  In  such 
a  case  it  is  well  to  modify  the  staining  method.  A  drop  of 
stain  is  placed  on  a  slide,  and  the  cover-glass,  film-side  down, 
lowered  upon  it.  After  the  lapse  of  the  time  necessary  for 
staining,  a  drop  of  water  is  placed  at  one  side 'of  the  cover-glass 
and  a  little  piece  of  filter-paper  at  the  other  side.  The  result  is 
that  the  stain  is  sucked  out  by  the  filter-paper.  By  adding 
fresh  drops  of  water  and  using  fresh  pieces  of  filter-paper,  the 
specimen  is  washed  without  any  violent  application  of  water, 
and  the  bacteria  are  not  displaced. 

For  the  general  staining  of  films  a  saturated  watery  solution 
of  methylene-blue  will  be  found  to  be  the  best  stain  to  com- 
mence with,  the  Gram  method  (vide  infra)  is  also  used,  and 
subsequently  any  special  stains  which  may  appear  advisable. 

The  Use  of  Mordants  and  Decolorising  Agents. — In  films 
of  blood  and  pus,  and  still  more  so  in  sections  of  tissues,  if  the 
above  methods  are  used,  the  tissue  elements  may  be  stained  to 
such  an  extent  as  to  quite  obscure  the  bacteria.  Hence  many 
methods  have  been  devised  in  which  the  general  principle  may 
be  said  to  be  (a)  the  use  of  substances  which,  while  increasing 
the  staining  power,  tend  to  fix  the  stain  in  the  bacteria,  and 
(6)  the  subsequent  treatment  by  substances  which  decolorise  the 
overstained  tissues  to  a  greater  or  less  extent,  while  they  leave 
the  bacteria  coloured.  The  staining  capacity  of  a  solution  may 
be  increased — 

(a)  By   the   addition   of   substances   such   as    carbolic  acid, 
aniline  oil,  or  metallic  salts. 

(b)  By  the  addition  of   alkalies,   such    as    caustic  potash  or 
ammonium  carbonate,  in  weak  solution. 

(c)  By  the  employment  of  heat. 

(d)  By  long  duration  of  the  staining  process. 


104  MICROSCOPIC  METHODS 

As  decolorising  agents  we  use  chiefly  mineral  acids  (hydro- 
chloric, nitric,  sulphuric),  vegetable  acids  (especially  acetic  acid), 
alcohol  (either  methylated  spirit  or  absolute  alcohol),  or  a  com- 
bination of  spirit  and  acid,  e.g.  methylated  spirit  with  a  drop  or 
two  of  hydrochloric  acid  added,  also  various  oils,  e.g.  aniline, 
clove,  etc.  In  most  cases  about  thirty  drops  of  acetic  acid  in 
a  bowl  of  water  will  be  sufficient  to  remove  the  excess  of  stain 
from  over-stained  films  and  sections.  More  of  the  acid  may,  of 
course,  be  added  if  necessary. 

Hot  water  also  decolorises  to  a  certain  extent ;  over-stained 
films  can  often  be  readily  decolorised  by  placing  a  drop  of  water 
on  the  film  and  heating  gently  over  a  flame. 

When  preparations  have  been  sufficiently  decolorised  by  an 
acid,  they  should  be  well  washed  in  tap  water,  or  in  distilled 
water  with  a  little  lithium  carbonate  added. 

Different  organisms  take  up  and  retain  the  stains  with  various 
degrees  of  intensity,  and  thus  duration  of  staining  and  decoloris- 
ing must  be  modified  accordingly.  We  sometimes  have  to  deal 
with  bacteria  which  show  a  special  tendency  to  be  decolorised. 
This  tendency  can  be  obviated  by  adding  a  little  of  the  stain  to 
the  alcohol,  or  aniline  oil,  employed  in  dehydration.  In  the 
latter  case  a  little  of  the  stain  is  rubbed  down  in  the  oil.  The 
mixture  is  allowed  to  stand.  After  a  little  time  a  clear  layer 
forms  on  the  top  with  stain  in  solution,  and  this  can  be  drawn 
off  with  a  pipette. 

When  methylene-blue,  methyl-violet,  or  gentian-violet  is  used, 
the  stain  can,  after  the  proper  degree  of  decolorisation  has  been 
reached,  be  fixed  in  the  tissues  by  treating  for  a  minute  with 
ammonium  molybdate  (2J  per  cent,  in  water). 

The  Formulae  of  some  of  the  more  commonly  used  Stain  Combinations. 

1.  Lojfler's  Methylene-blue. 

Saturated  solution  of  methylene-blue  in  alcohol        .         .         .         30  o.c. 
Solution  of  potassium  hydrate  in  distilled  water  (1-10,000)      .       100    ,, 

(This  dilute  solution  maybe  conveniently  made  by  adding  1  c.c.  of  a 
1  per  cent,  solution  to  99  c.c.  of  water. ) 

Sections  may  be  stained  in  this  mixture  for  from  a  quarter  of  an  hour 
to  several  hours.  They  do  not  readily  overstain.  The  tissue  containing 
the  bacteria  is  then  decolorised  if  necessary  with  ^-1  per  cent,  acetic  acid, 
till  it  is  a  pale  blue-green.  The  section  is  washed  in  water,  rapidly 
dehydrated  with  alcohol  or  aniline  oil,  cleared  in  xylol,  and  mounted. 

The  tissue  may  be  contrast-stained  with  eosin.  If  this  is  desired, 
after  decolorisation  wash  with  water,  place  for  a  few  seconds  in  1  per 
cent,  solution  of  eosin  in  absolute  alcohol,  rapidly  complete  dehydration 
with  pure  absolute  alcohol,  and  proceed  as  before. 


GRAM'S  STAIN  105 

Films  may  l>e  stained  with  Loflier's  blue  by  five  minutes'  exposure  or 
longer  in  the  cold.  They  usually  do  not  require  decolorisation,  as  the 
tissue  elements  are  not  overstained. 

2.  Kiihnts  Methylene-bluc. 

Methylene-blue         ....         l'5grm. 
Absolute  alcohol       ....       10  c.c. 
Carbolic  acid  solution  (1-20)    .         .     100   ,, 

Stain  and  decolorise  as  with  Loffler's  blue,  or  decolorise  with  very  weak 
hydrochloric  acid  (a  few  drops  in  a  bowl  of  water). 

3.  Carbol'Thionin-blue.—^lakc  up  a  stock   solution   consisting  of  1 
gramme  of  thionin-blue  dissolved  in  100  c.c.  carbolic  acid  solution  (1-40). 
For  use,  dilute  one  volume  with  three  of  water,  and  filter.     Stain  sections 
for  five  minutes  or  upwards.     Wash  very  thoroughly  with  water,  other- 
wise a  deposit  of  crystals  may  occur  in  the  subsequent  stages.     Decolorise 
with  very  weak  acetic  acid.     A  few  drops  of  the  acid  added  to  a  bowl 
of  water    are  quite    sufficient.     Wash    again    thoroughly  with   water. 
Dehydrate   with    absolute    alcohol.     Thionin-blue    stains    more   deeply 
than  methylene-blue,  and  gives  equally  good  differentiation.     It  is  very 
suitable  for  staining  typhoid  and  glanders  bacilli  in  sections.     Cover- 
glass  preparations    stained    by   this    method    do    not    usually  require 
decolorisation.     As  a  contrast  stain,  1  per  cent,  watery  solution  of  eosin 
may  be  used  before  staining  with  the  thionin. 

4.  Gentian-violet  in  Aniline  Oil   Water. — Two  solutions  have  here  to 
be   made  up.     (a)  Aniline   oil  water.     Add  about  5  c.c.  aniline  oil  to 
100  c.c.  distilled  water  in  a  flask,  and  shake  violently  till  as  much  as 
possible  of  the  oil  has  dissolved.     Filter  and  keep  in  a  covered  bottle 
to  prevent  access  of  light.     (6)  Make  a   saturated   solution  of  gentian- 
vi'.l.-t  in  alcohol.     When  the  stain  is  to  be  used,  1  part  of  (b)  is  added 
to  10  parts  of  (a),  and  the  mixture  filtered.     The  mixture  should  be  made 
not  more  than  twenty- four  hours  before  use.     Stain  sections  for  a  few 
minutes ;    then    decolorise   with    methylated    spirit.     Sometimes    it  is 
advantageous  to  add  to  the  methylated  spirit  a  little  hydrochloric  acid 
(2-3  minims  to  100  c.c.).     This  staining  solution  is  not  so  much  used 
by  itself  as  in  <  J ram's  method,  which  is  presently  to  be  described. 

5.  Carbol-Gentian-Violet. — 1    part  of  saturated   alcoholic  solution  of 
gentian-violet  is  mixed  with  10  parts  of  5  per  cent,  solution  of  carbolic 
acid.     It  is  used  as  No.  4. 

6.  Carbol-Fuchsin  (see  p.  1C8). — This  is  a  very  powerful  stain,  and, 
when  used  in  the  undiluted  condition,  £-1  minute's  staining  is  usually 
sufficient.     It  is  better,  however,  to  dilute  with  from  five  to  ten  times 
its  volume  of  water  and  stain  for  a  few  minutes.     In  this  form  it  has  a 
very  wide  application.     Methylated  spirit  with  or  without  a  few  drops 
of  acetic  acia  is  the  most  convenient  decolorising  agent.     Then  dehydrate 
thoroughly,  clear,  and  mount. 

Gram's  Method  and  its  Modifications. — In  the  methods 
alivady  described,  the  tissues,  and  more  especially  the  nuclei, 
ivt;iiu  some  stain  when  decolorisation  has  reached  the  }>oint  to 
which  it  can  safely  go  without  the  bacteria  themselves  bein^ 
affected.  In  the  method  of  Gram,  now  to  be  detailed,  this  does 
not  occur,  for  the  stain  can  here  be  removed  completely  from 


106  MICROSCOPIC  METHODS 

the  ordinary  tissues,  and  left  only  in  the  bacteria.  All  kinds 
of  bacteria,  however,  do  not  retain  the  stain  in  this  method, 
and  therefore  in  the  systematic  description  of  any  species  it  is 
customary  to  state  whether  it  is,  or  is  not,  stained  by  Gram's 
method — by  this  is  meant,  as  will  be  understood  from  what 
has  been  said,  whether  the  particular  organism  retains  the 
colour  after  the  latter  has  been  completely  removed  from  the 
tissues.  It  must,  however,  be  remarked  that  some  tissue 
elements  may  retain  the  stain  as  firmly  as  any  bacteria,  e.g. 
keratinised  epithelium,  calcified  particles,  the  granules  of  mast 
cells,  and  sometimes  altered  red  blood  corpuscles,  etc. 

In  Gram's  method  the  essential  feature  is  the  treating  of  the 
tissue,  after  staining,  with  a  solution  of  iodine.  This  solution 
is  spoken  of  as  Gram's  solution,  and  has  the  following  com- 
position : — 

Iodine         .          .          .          .          .          1  part. 
Potassium  iodide          ...          2  parts. 

Distilled  water    ....     300      „ 

• 

The  following  is  the  method  : — 

1.  Stain  in  aniline  oil  gentian -violet  or  in  carbol-gentian- violet  (vide 
supra),  for  about  five  minutes. 

2.  Without  washing   in    water,  now   treat   the   section   or   film  with 
repeated   doses   of  Gram's  solution   till   its   colour   becomes  a  purplish 
black,  and  allow  the  solution  to  net  for  one  minute. 

3.  Again  without  washing  with  water,  decolorise  with  absolute  alcohol 
or  methylated  spirit  till  the  colour  has  almost  entirely  disappeared,  the 
tissues  having  only  a  faint  violet  tint.     Tlie  period  of  time  tor  which  the 
alcohol  is  allowed  to  act  varies  in  different  laboratories.     The  best  period 
is  probably  about  three  minutes. 

4.  Dehydrate  completely,  clear  with  xylol,  and  mount.     In  the  case 
of  film  preparations,  the  specimen  is  simply  washed  in  water,  dried,  and 
mounted. 

In  stage  (3)  the  process  of  decolorisation  is  more  satisfactorily  per- 
formed by  using  clove  oil  after  sufficient  dehydration  with  alcohol,  the 
clove  oil  being  afterwards  removed  by  xylol. 

As  a  contrast  stain  for  the  tissues,  carmalum  or  lithia  carmine  is  used 
before  staining  with  gentian-violet  (1).  As  a  contrast  stain  for  other 
bacteria  which  are  decolorised  by  Gram's  method,  carbol-fuchsin  diluted 
with  twenty  volumes  of  water  or  a  saturated  watery  solution  of  Bismarck- 
brown  may  be  used  before  stage  (4) ;  the  former  should  not  be  applied 
for  longer  than  a  few  seconds. 

The  following  modifications  of  Gram's  method  may  be  given  : — 

1.  Weigert's  Modification. — The  contrast  staining  of  the  tissues  and 
stages  (1)  and  (2)  are  performed  as  above. 

(3)  After  using  the  iodine  solution  the  preparation  is  dried  by  blotting 
and  then  decolorised  by  aniline-xylol  (aniline-oil  2,  xylol  1). 


TUBERCLE  STAINS  107 

(4)  Wash  well  in  xylol,  and  mount  in  xylol  balsam.  Film  preparations 
after  being  washed  in  xylol  may  be  dried,  and  thereafter  dilute  carbol- 
fuchsin  may  be  used  to  stain  bacteria  which  have  been  decolorised. 

This  modification  probably  gives  the  most  uniformly  successful  results. 

2.  Nicolle's  Modification. — Carbol-gentian-violet  is  used  as  the  stain. 
Treatment  with  iodine  is  carried  out  as  above,  and  decolorisation   is 
effected  with  a  mixture  of  acetone  (1  part)  and  alcohol  (2  parts). 

3.  Kiihnes  Modification. — (1)   Stain   for  five  minutes  in   a   solution 
made  up  of  equal  parts  of  saturated  alcoholic  solution  of  crystal-violet 
("  Krystall- violet  ")  and  1  per  cent,  solution  of  ammonium  carbonate. 

(2)  Wash  in  water. 

(3)  Place  for  two  to   three  minutes  in  Gram's  iodine  solution,  or  in 
the  following  modification  by  Kiihne  : — 

Iodine 2  parts. 

Potassium  iodide        .         .         .         .  4     ,, 

Distilled  water  .         .         .         .         .       100     ,, 

For  use,  dilute  with  water  to  make  a  sherry- coloured  solution. 

(4)  Wash  in  water. 

(5)  Decolorise   in    a    saturated    alcoholic    solution    of    fluorescein   (a 
saturated  solution  in  methylated  spirit  does  equally  well). 

(6)  Dehydrate,  clear,  and  mount. 

There  is  great  variability  in  the  avidity  with  which  organisms  stained 
by  Gram  retain  the  dye  when  washed  with  alcohol,  and  sometimes 
difficulty  is  experienced  in  saying  whether  an  organism  does  or  does  not 
stain  by  this  method. 

Most  bacteria  are  either  frankly  Gram-positive  or  frankly 
Gram-negative,  but  cases  occur  when  an  organism,  usually  Gram- 
positive  or  Gram-negative,  tends  when  grown  on  certain  media  to 
show  an  opposite  tendency,  and  sometimes  an  organism  is  met 
with  in  which  the  individuals  in  a  film  show  slightly  different 
reactions  to  the  Gram  stains. 

Stain  for  Tubercle  and  other  Acid-fast  Bacilli. — These 
bacilli  cannot  be  well  stained  with  a  simple  watery  solution  of 
a  basic  aniline  dye.  This  fact  can  easily  be  tested  by  at- 
tempting to  stain  a  film  of  a  tubercle  culture  with  such  a 
solution ;  with  the  Gram  method,  however,  a  partial  staining 
is  sometimes  effected.  Such  bacteria  require  a  powerful  stain 
containing  a  mordant,  and  must  be  exposed  to  the  stain  for  a 
long  time,  or  its  action  may  be  aided  by  a  short  application  of 
heat.  When  once  stained,  however,  they  resist  decolorising 
even  with  very  powerful  acids;  they  are  therefore  called  "acid- 
fast."  The  smegma  bacillus  also  resists  decolorising  with 
strong  acids  (p.  280),  and  a  considerable  number  of  other  acid- 
fast  bacilli  are  now  known  (p.  278).  Any  combination  of 
gentian-violet  or  fuchsin  with  aniline  oil  or  carbolic  acid  or 


108  MICROSCOPIC  METHODS 

other  mordant  will  stain  the  bacilli  named,  but  the  following 
methods  are  most  commonly  used  : — 

Ziehl-Neelsen  Carbol-Fuchsin  Stain. 

Basic  fuchsin    .  .          .  1  part. 

Absolute  alcohol        .         .         .         .          10  parts. 
Solution  of  carbolic  acid  (1  :  20)         .        100     „ 

1.  Place  the  specimen  in  this  fluid,  and  having  heated  it  till  steam 
rises,  allow  it  to  remain  there  for  five  minutes,  or  allow  it  to  remain  in 
the  cold  stain  for  from  twelve  to  twenty-four  hours.     (Films  and  paraffin 
sections  are  usually  stained  with  hot  stain,  loose  sections  with  cold  ;  in 
hot  stain  the  latter  shrink.) 

2.  Decolorise  with  20  per  cent,  solution  of  strong  sulphuric  acid,  nitric 
acid,  or  hydrochloric  acid,  in  water.     In  this  the  tissues  become  yellow. 

3.  Wash  well  with  water.     The  tissues  will  regain  a  faint  pink  tint. 
If  the  colour  is  distinctly  red,  the  decolorisation  is  insufficient,  and  the 
specimen  must  be  returned  to  the  acid.     As  a  matter  of  practice,  it  is 
best   to   remove   the  preparation    from   the   acid  every  few  seconds  and 
wash  in  water,  replacing  the  specimen  in  the  acid  and  re-washing  till 
the  proper  pale  pink  tint  is  obtained.     Then  wash  in  alcohol  for  half  a 
minute,  and  replace  in  water. 

4.  Contrast  stain  with  a  saturated  watery  solution  of  methyleue-blue 
for  half  a  minute,   or  with  saturated  watery  Bismarck-brown  for  from 
two  to  three  minutes. 

5.  Wash  well  with  water.     In  the  case  of  films,  dry  and  mount.     In 
the  case  of  sections,  dehydrate,  clear,  and  mount. 

Fraenkel's  Modification  of  the  Ziehl-Neelsen  Stain. 

Here  the  process  is  shortened  by  using  a  mixture  containing 
both  the  decolorising  agent  and  the  contrast  stain. 

The  sections  or  films  are  stained  with  the  carbol-fuchsin  as  above 
described,  and  then  placed  in  the  following  solution  : — 

Distilled  water   ......  50  parts. 

Absolute  alcohol 30      ,, 

Nitric  acid .         .         .         .  .  20     ,, 

Methylene-blue  in  crystals  to  saturation. 

They  are  treated  with  this  till  the  red  colour  has  quite  disappeared  and 
been  replaced  by  blue.  The  subsequent  stages  are  the  same  as  in  No.  5, 
supra. 

Leprosy  bacilli  are  stained  in  the  same  way,  but  are  rather 
more  easily  decolorised  than  tubercle  bacilli,  and  it  is  better 
to  use  only  5  per  cent,  sulphuric  acid  in  decolorising. 

In  the  case  of  specimens  stained  either  by  the  original  Ziehl- 
Neelsen  method,  or  by  Fraenkel's  modification,  the  tubercle  or 


STAINING  OF  SPORES  AND  CAPSULES        109 

leprosy  bacilli  ought  to  be  bright  red,  and  the  tissue  blue  or 
In-own,  according  to  the  contrast  stain  used.  Other  bacteria 
which  may  be  present  are  also  coloured  with  the  contrast  stain. 

The  Staining  of  Spores. — If  bacilli  containing  spores  are 
stained  with  a  watery  solution  of  a  basic  aniline  dye  the  spores 
remain  unstained.  The  spores  either  take  up  the  stain  less 
readily  than  the  protoplasm  of  the  bacilli,  or  they  have  a  resisting 
envelope  which  prevents  the  stain  penetrating  to  the  protoplasm. 
Like  the  tubercle  bacilli,  when  once  stained  they  retain  the 
colour  with  considerable  tenacity.  The  following  is  the  simplest 
method  for  staining  spores  : — 

1.  Stain  cover-glass  films  as  for  tubercle  bacilli. 

2.  Decolorise  with  1  per  cent,  sulphuric  acid  in  water  or  with  methy- 
lated spirit.     This  removes  the  stain  from  the  bacilli. 

3.  Wash  in  water. 

4.  Stain  with  saturated  watery  methylene-blue  for  half  a  minute. 

5.  Wash  in  water,  dry,  and  mount  in  balsam. 

The  result  is  that  the  spores  are  stained  red,  the  protoplasm  of  the 
bacilli  blue. 

The  spores  of  some  organisms  lose  the  stain  more  readily  than  those 
of  others,  and  for  some,  methylated  spirit  is  a  sufficiently  strong 
decolorising  agent  for  use.  If  sulphuric  acid  stronger  than  1  per  cent, 
is  used,  the  spores  of  many  bacilli  are  readily  decolorised. 

Mt,1t<-rx  M'tluul.—  The  following  method,  recommended  by  Moller,  is 
much  more  satisfactory  than  the  previous.  Before  being  stained,  the 
films  are  placed  in  chloroform  for  two  minutes,  and  then  in  a  5  per  cent, 
solution  of  chromic  acid  for  ^-2  minutes,  the  preparation  being  well 
washed  after  each  reagent.  Thereafter  they  are  stained  and  decolorised 
as  above. 

The  Staining  of  Capsules. — The  two  following  methods  may 
be  recommended  in  the  case  of  capsulated  bacteria  : — 

(a)  Welch's  Method. — This  depends  on  the   fact  that   in  many  cases 
the  capsules  can  be  fixed  with  glacial  acetic  acid. 

Films  when  still  wet  are  placed  in  this  acid  for  a  few  seconds. 

The  superfluous  acid  is  removed  with  filter-paper,  and  the  preparation 
is  treated  with  gentian-violet  in  aniline  oil  water  repeatedly  till  all  the 
acetic  acid  is  removed. 

Then  wash  with  1-2  per  cent,  solution  of  sodium  chloride,  and  examine 
in  the  same  solution. 

The  capsule  appears  as  a  pale  violet  halo  around  the  deeply  stained 
bacterium. 

(b)  Hiss's  Method.—  The  staining  solution   consists  of  1   part  of  a 
saturated  alcoholic  solution  of  fuchsin  or  gentian-violet  and  19  parts  of 
distilled  water.     A  few  drops  of  the  stain  are  placed  on  a  film,  previously 
<lri«'(l  and  fixed  by  heat,  and  the  preparation  is  steamed  for  a  few  seconds  over 
a  flame.     The  staining  solution  is  washed  off  with  a  20  per  cent,  solution 
of  copper  sulphate,  the  preparation  (without  being  washed  in  water)  is  dried 
between  filter-papers,  and  when  thoroughly  dry  is  mounted  in  balsam.    The 
capsules  of  pneumococci  growing  in  a  fluid  serum  medium  can  be  readily 


110  MICROSCOPIC  METHODS 

demonstrated  by  this  method  ;  in  the  case  of  solid  cultures  films  should 
be  made  without  any  diluent,  or  a  drop  of  fluid  serum  should  be  used. 
The  method  is  easily  applied,  and  gives  excellent  results. 
(c)  Richard  Muir's  Method  (as  recently  modified). 

1.  The  film  containing  the  bacteria  must  be  very  thin.     It  is  dried 
and  stained  in  filtered  carbol-fuchsin  for  half  a  minute,  the  preparation 
being  gently  heated. 

2.  Wash  slightly  with  spirit  and  then  well  in  water. 

3.  Place  in  following  mordant  for  a  few  seconds  : — 

Saturated  solution  of  corrosive  sublimate .         .         .2  parts. 
Tannic  acid  solution — 20  per  cent.    .         .         .         .     2     , , 
Saturated  solution  of  potash  alum    .         .         .         .     5     , , 

4.  Wash  well  in  water. 

5.  Treat  with  methylated  spirit  for  about  a  minute. 
The  preparation  has  a  pale  reddish  appearance. 

6.  Wash  well  in  water. 

7.  Counterstain  with  watery  solution  of  ordinary  methyl ene-blue  for 
half  a  minute. 

8.  Dehydrate  in  alcohol,  clear  in  xylol,  and  mount  in  balsam. 

The  bacteria  are  a  deep  crimson,  and  the  capsules  of  a  blue  tint.  The 
capsules  of  bacteria  in  certain  culture  media  may  be  demonstrated  by 
this  method. 

The  Staining  of  Flagella. — The  staining  of  the  flagella  of 
bacteria  is  the  most  difficult  of  all  bacteriological  procedures, 
and  it  requires  considerable  practice  to  ensure  that  good  results 
shall  be  obtained.  Many  methods  have  been  introduced,  of 
which  the  two  following  are  the  most  satisfactory  : — 

Preparation  of  Films.  —  In  all  the  methods  of  staining 
flagella,  young  cultures  on  agar  should  be  used,  say  a  culture 
incubated  for  from  ten  to  eighteen  hours  at  37°  C.  A  very 
small  portion  of  the  growth  is  taken  on  the  point  of  a  platinum 
needle,  and  carefully  mixed  in  a  little  water  in  a  watch-glass ; 
the  amount  should  be  such  as  to  produce  scarcely  any  turbidity 
in  the  water.  A  film  is  then  made  by  placing  a  drop  on  a 
clean  cover-glass  and  carefully  spreading  it  out  with  the  needle. 
It  is  allowed  to  dry  in  the  air,  and  is  then  passed  twice  or  thrice 
through  a  flame,  care  being  taken  not  to  over-heat  it.  The 
cover-glasses  used  should  always  be  cleaned  in  the  mixture  of 
sulphuric  acid  and  potassium  bichromate  described  on  page  93. 

1.  PitfieWs  Method  as  modified  by  Richard  Muir. 

Prepare  the  following  solutions  : — 
A.   The  Mordant. 

Tannic  acid,  10  per  cent,  watery  solution,  filtered     .  10  c.c. 

Corrosive  sublimate,  saturated  watery  solution          .  5    ,, 

Alum,  saturated  watery  solution        .         .         .  5    ,, 

Carbol-fuchsin  (vide  p.  108) 5    ,, 


STAINING  OF  FLAGELLA  111 

Mix  thoroughly.  A  precipitate  forms,  which  must  be  allowed  to 
deposit,  either  by  centrifugalising  or  simply  by  allowing  to  stand. 
Remove  the  clear  fluid  with  a  pipette,  and  transfer  to  a  clean  bottle. 
The  mordant  keeps  well  for  one  or  two  weeks. 

B.   The  Stain. 

Alum,  saturated  watery  solution     .         .         .         .     10  c.c. 
Gentian- violet,  saturated  alcoholic  solution     .         .       2    ,, 

The  stain  should  not  be  more  than  two  or  three  days  old  when  used. 
It  may  be  substituted  in  the  mordant  in  place  of  the  carbol-fuchsin. 

The  film  having  been  prepared  as  above  described,  pour  over  it  as 
much  of  the  mordant  as  the  cover-glass  will  hold.  Heat  gently  over  a 
Hume  till  steam  begins  to  rise,  allow  to  steam  for  about  a  minute,  and 
then  wash  well  in  a  stream  of  running  water  for  about  two  minutes. 
Then  dry  carefully  over  the  flame,  and  when  thoroughly  dry  pour  on 
some  of  the  stain.  Heat  as  before,  allowing  to  steam  for  about  a  minute, 
wash  well  in  water,  dry,  and  mount  in  a  drop  of  xylol  balsam. 

This  method  has  yielded  the  best  results  in  our  hands. 

2.   Van  Ermengeni's  Method  for  Staining  Flagella. 

The  films  are  prepared  as  above  described.  Three  solutions  are  here 
necessary  : — 

Solution  A.     (Bain  fixatcur)— 

Osmir  acid,  2  per  cent,  solution          ...         1  part. 
Tannin,  10-25  per  cent,  solution        ...         2  parts. 

Place  the  films  in  this  for  one  hour  at  room  temperature,  or  heat  over 
a  flame  till  steam  rises  and  keep  in  the  hot  stain  for  five  minutes. 
Wash  with  distilled  water,  then  with  absolute  alcohol  for  three  to  four 
minutes,  and  again  in  distilled  water,  and  treat  with 

Solution  B.  (Bain  sensibilisaleur] — 

•5  per  cent,  solution  of  nitrate  of  silver  in  distilled  water.  Allow 
films  to  be  in  this  a  few  seconds.  Then  without  washing  transfer  to 

Solution  C.     (Bain  reducteur  et  reinforfateur) — 

Gallic  acid          .......         5  grms. 

Tannin      ........         3     ,, 

Fused  potassium  acetate 10    ,, 

Distilled  water 350  c.c. 

Keep  in  this  for  a  few  seconds.  Then  treat  again  with  Solution  B  till 
the  preparation  begins  to  turn  black.  Wash,  dry,  and  mount. 

It  is  better,  as  Mervyn  Gordon  recommends,  to  leave  the  specimen  in 
B  for  two  minutes,  and  then  to  transfer  to  C  for  one  and  a  half  to  two 
minutes,  and  not  to  transfer  again  to  B.  It  will  also  be  found  an 
advantage  to  use  a  fresh  supply  of  C  for  each  preparation,  a  small 
quantity  being  sufficient.  The  beginner  will  find  the  typhoid  bacillus 
or  the  bacillus  coli  communis  very  suitable  organisms  to  stain  by  this 
method. 


112  MICROSCOPIC  METHODS 

Although  the  results  obtained  by  this  method  are  sometimes  excellent, 
they  vary  considerably.  Frequently  both  the  organisms  and  flagella 
appear  of  abnormal  thickness.  This  is  due  to  the  fact  that  the  process 
on  which  the  method  depends  is  a  precipitation  rather  than  a  true 
staining.  The  pictures  on  the  whole  are  less  faithful  than  in  the  first 
method. 

Staining  of  Spirochsetes  in  Sections. — The  following  im- 
pregnation methods  have  been  applied  for  this  purpose  by 
Levaditi,  and  give  excellent  results : — 

(a)  Levaditi's  Original  Method. 

(1)  The  tissues,  which  ought  to  be  in  thin  slices,   about  1  mm.  in 
thickness,  are  best  fixed  in  10  per  cent,  formalin  solution  for  twenty-four 
hours. 

(2)  They  are  washed  for  an  hour  in  water,  and  then  brought  into  96  per 
cent,  alcohol  for  twenty-four  hours. 

(3)  They  are  then  placed  in  1 -5  per  cent,  solution  of  nitrate  of  silver  in 
a  dark  bottle,  and  are  kept  in  an  incubator  at  37°  C.  for  three  days. 

(4)  They   are  washed   in   water   for   about  twenty  minutes,  and  are 
thereafter  placed  in  the  following  mixture,  namely  : — 

Pyrogallic  acid,  4  parts. 

Formalin,  5  parts. 

Distilled  water  up  to  100  parts. 

They  are  kept  in  this  mixture  in  a  dark  bottle  for  forty-eight  hours  at 
room  temperature. 

(5)  They  are  then  washed  in  water  for  a  few  minutes,  taken  through 
increasing  strengths  of  alcohol,  and  embedded  in  paraffin  in  the  usual 
way.     The  sections  ought   to   be   as   thin   as  possible.     In  satisfactory 
preparations  the  spirochsetes  appear  of  an  almost  black  colour  against  the 
pale   yellow  background  of  the   tissues.     The  latter  can   be   contrast- 
stained  by  weak  carbol- fuchsin  or  by  toluidin  blue. 

(b)  Levaditi's  Newer  Pyridin  Method. 

(1)  The  tissues  are  fixed  in  formalin  as  in  the  previous  method,  are 
hardened  in  alcohol  for  twelve  to  sixteen  hours,  and  then  washed  in  water. 

(2)  They  are  then  impregnated  with  a  1   per  cent,   solution  of  silver 
nitrate,  to  which  10  per  cent,  of  pyridin  puriss.  is  added  at  the  time  of 
use.     The  tissues  are  placed  in  the  solution  in  a  well-stoppered  bottle,  and 
are  kept  for  two  to  three  hours  at  room  temperature  and  four  to  six  hours 
at  about  50°  C.     They   are  thereafter  washed  quickly  in  10  per  cent, 
pyridin  solution. 

(3)  Reduction  is  then  carried  out  in  the  following  mixture,  namely,  a 
4  per  cent,  solution  of  pyrogallic  acid  to  which  are  added,  at  the  time  of 
use,  10  per  cent,  pure  acetone  and  15  per  cent,  pyridin. 

(4)  The  tissues  are  then  put  through  alcohol  and  xylol,  and  embedded 
in  paraffin.     The  sections  can  be  stained  with  toluidin  blue  or  Unna's 
polychrome  blue. 

(For  the  staining  of  spirochoetes  in  films,  see  p.  115.) 


THE  ROMANOWSKY  STAINS  113 

The  Romanowsky  Stains. — Within  recent  years  the  numerous 
mollifications  of  the  Romanowsky  stain  have  been  extensively 
used.  The  dye  concerned  is  the  compound  which  is  formed 
when  watery  solutions  of  medicinal  methylene-blue  and  water- 
soluble  eosin  are  brought  together.  This  compound  is  insoluble 
in  water  but  soluble  in  alcohol — the  alcohol  employed  being 
methyl  alcohol.-  The  stain  was  originally  used  by  Romanowsky 
for  the  malarial  parasite,  and  its  special  quality  is  that  it 
imparts  to  certain  elements,  such  as  the  chromatin  of  this 
oruiiiiHin,  ;i  reddish-purple  hue.  This  was  at  first  thought  to  be 
simply  due  to  the  combination  of  the  methylene-blue  and  the 
eosin,  but  it  is  now  recognised  that  certain  changes,  such  as 
occur  in  methylene-blue  solutions  with  age,  are  necessary.  In 
the  modern  formula;  these  changes  are  brought  about  by 
treatment  with  alkalies,  especially  alkaline  carbonates,  as  was 
first  practised  by  Unna  in  the  preparation  of  his  polychrome 
methylene-blue.  The  stains  in  use  thus  contain  a  mixture  of 
methylene-blue  and  its  derivatives  in  combination  with  eosin ; 
the  differences  in  these  bodies  and  the  different  proportions  in 
which  they  occur  in  individual  stains  account  for  the  different 
effects  produced  on  the  various  constituents  of  a  cell.  The 
underlying  chemical  reactions  are  complicated  and  as  yet  not 
fully  understood.  Thus  it  is  not  certainly  known  to  what  partic- 
ular new  body  the  reddish  hue  produced  in  chromatin  is  due, 
but  the  active  constituent  may  be  methyl-violet  or  methyl-azure 
or  thionin,  all  of  which  result  from  the 'action  of  alkali  on 
methylene-blue.  The  stains  are  much  used  in  staining  blood- 
lilms  (in  which  the  characters  of  both  nucleus  and  cytoplasm 
in  leucocytes  are  beautifully  brought  out),  in  staining  bacteria 
in  tissues  or  exudates,  the  malaria  parasite,  trypanosomes,  the 
pathogenic  spirochajtes  (such  as  the  spirochaete  pallida),  and 
protozoa  generally. 

The  following  are  the  chief  formulae  in  use  : — 

1.  Jcnner's  Stain. — This  is  an  excellent  blood  stain,  hut  is  not  so  good 
for  the  study  of  parasites  as  the  others  to  be  mentioned.  In  its 
preparation  n«>  alkali  is  used.  It  is  made  by  mixing  equal  parts  of  (a) 
a  1'2  to  1*25  per  cent,  solution  of  Griibler's  water-soluble  eosin  (yellow 
shade)  in  distilled  water  and  (b)  1  per  cent.  Griibler's  medicinal  methy- 
leue-blue  (also  a  watery  solution).  The  mixture  is  allowed  to  stand 
twenty- four  hours,  is  filtered,  and  the  residue  is  dried  at  55°  C.  ;  the 
powder  is  shaken  up  in  distilled  water,  filtered,  washed  with  distilled 
wat«-r,  and  dried.  Of  the  powder,  '5  grin,  is  dissolved  in  100  c.c.  Merck's 
methyl  alcohol.  For  use  a  few  drops  are  placed  on  the  dried  uniixed 
liliu  for  one  to  three  minutes,  the  dye  is  poured  off,  and  the  pre  pa  ration 
wa.-Oied  with  distilled  water  till  it  presents  a  pink  colour;  it  is  then 
dried  between  (liter-paper  and  mounted  in  xylol  balsam. 

8 


114  MICROSCOPIC  METHODS 

2.  Leishman's  Stain. — The  following  solutions  are  prepared  :    (a)  to 
a  1  per  cent,  solution  of  medicinal  methylene-blue  is  added  "5  per  cent, 
sodium  carbonate  ;  the  mixture  is  kept  at  65°  C.  for  twelve  hours,  and 
then  for  ten  days  at  room  temperature  ('25  per  cent,   formalin  may  be 
added  as  a  preservative) ;   (&)  1-1000  solution  of  eosin,  extra  B.A.,  in 
distilled  water.      Equal  volumes  of  the   two  solutions  are  mixed  and 
allowed  to  stand  for  six  to  twelve   hours  with  occasional  stirring,   the 
precipitate  is  collected,  filtered,  washed  with  distilled  water,  and  dried. 
For  use,    '15   per  cent,    is  dissolved  in  Merck's  methyl  alcohol  ("for 
analysis,    acetone   free")    as   follows:    The  powder  is  placed  in  a   clean 
mortar,   a  little  of  the  alcohol   is  added  and  well  rubbed  up  with  a 
pestle ;    the   undissolved   powder   is   allowed    to    settle    and    the    fluid 
decanted  into  a  dry  bottle ;  the  process  is  repeated  with  fresh  fractions 
of  the  solvent  till  practically  all  the  stain  is  dissolved,  and  the  bottle 
is  well   stoppered.     The  stain  will   keep   for  a  long   period.     For   the 
staining  of  films  a  few  drops  of  the  stain  are  placed   on  the  unfixed 
preparation    for    fifteen    to    thirty   seconds  so  as  to   cover   it  with   a 
shallow  layer   (the   stain   may  be    conveniently   spread   over  the   film 
with  a  glass  rod),  and  the  film  is  tilted  to  and  fro  so  as  to  prevent 
drying.     This  treatment  efficiently  fixes  the  film  by  the  action  of  the 
methyl  alcohol.     About  double  the  quantity  of  distilled  water  is  now 
dropped  on  the  film,  and  the  stain  and  diluent  are  quickly  mixed  with 
the  rod.     Five  minutes  are  now  allowed  for  staining,  and  the  stain  is 
then  gently  washed  off  with  distilled  water.     A  little  of  the  water  is 
kept  on  the  film  for  half  a  minute  to  intensify  the  colour  contrasts  in 
the  various  cells.     For  certain  special  structures,  such  as  Schuffner's  dots 
or  Maurer's  dots  in  the  malarial  parasite,  a  longer  staining  (up  to  one 
hour)  may  be  necessary,  and  in  any  case  it  is  well  to  practise  being  able 
to  control  the  depth  of  the  staining  effect  by  observation  with  a  low- 
power   objective.     If  a  preparation  is  to  be  stained  for  a  long  time  it 
must  be  kept  covered,  and  if  in  such  cases  a  granular  deposit  is  formed 
this  may  be  got  rid  of  by  a  quick  wash  with  absolute  alcohol.     If  in  blood 
films  the  red  corpuscles  appear  bluish  instead  of  pink,  the  colour  may 
be  restored  by  washing  the  film  with  acetic  acid,  1-1500.     The  film  is 
dried  between  filter-paper  and  mounted. 

For  staining  sections  a  little  modification  is  necessary.  A  paraffin 
section  is  taken  into  distilled  water  as  usual,  the  excess  of  water  is  drained 
off,  and  a  mixture  of  one  part  of  stain  and  two  parts  of  distilled  water 
is  placed  on  it.  The  stain  is  allowed  to  act  for  five  to  ten  minutes  till  the 
tissue  appears  a  deep  Oxford  blue  ;  it  is  then  decolorised  with  1-1500 
acetic  acid — the  effect  being  watched  under  a  low-power  lens.  The  blue 
begins  to  come  out,  and  the  process  is  allowed  to  go  on  till  only  the 
nuclei  remain  blue.  The  section  is  then  washed  with  distilled  water, 
rapidly  dehydrated  with  alcohol,  cleared,  and  mounted.  If,  as  some- 
times happens,  the  eosin  tint  be  too  well  marked,  it  can  be  lightened 
by  the  action  of  1-7000  solution  of  caustic  soda,  this  being  washed  off 
whenever  the  desired  colour  has  been  attained. 

In  certain  cases,  e.g.  for  the  staining  of  old  films  or  of  trypanosomes 
or  Leishmanife  in  sections,  Leishrnan  recommends  an  initial  treat- 
ment of  the  preparation  with  serum.  This  modification  is  described  in 
Appendix  E. 

3.  J.  II.   Wright's  Stain. — In  this  modification  1  per  cent,  methylene- 
blue  (BX  or  Ehrlich's  rectified)  and  ^  per  cent,  sodium  carbonate  (both 
in  water)  are  mixed  and  placed  in  a  Koch's  steriliser  for  an  hour.     When 


THE  ROMANOWSKY  STAINS  115 

the  fluid  is  cold,  1-1000  solution  of  extra  B.  A.  eosiii  is  added  till  the  mix- 
ture becomes  purplish  and  a  finely  granular  black  precipitate  appears  in 
suspension  (about  500  c.c.  eosin  to  100  c.c.  methylene-blue  solution  are 
required)  ;  the  precipitate  is  filtered  off  and  dried  without  being  washed. 
A  saturated  solution  of  this  is  made  in  the  pure  methyl  alcohol ;  this  is 
filtered  and  diluted  by  adding  to  80  c.c.  of  the  saturated  solution  20  c.c. 
of  methyl  alcohol.  The  application  of  the  stain  is  almost  the  same  as 
with  Leishman's.  A  few  drops  are  placed  on  the  preparation  for  a 
minute  for  fixation  ;  water  is  then  dropped  on  till  a  green  iridescent 
scum  appears  on  the  top  of  the  fluid,  and  staining  goes  on  for  about 
two  minutes ;  the  stain  is  then  washed  off  with  distilled  water,  and 
a  little  is  allowed  to  remain  on  the  film  till  differentiation  is  com- 
plete ;  the  preparation  is  carefully  dried  with  filter  -  paper,  and 
mounted. 

4.  Giemsa's  Stain.  —  Giemsa  believes  that  the  reddish-blue  hue 
characteristic  of  the  Romanowsky  stain  is  due  to  the  formation  of 
methyl-azure,  and  he  has  prepared  this  by  a  method  of  his  own  under 
the  name  "Azur  I."  From  this,  by  the  addition  of  equal  parts  of 
medicinal  methylene-blue,  he  prepares  what  he  calls  "Azur  II.,"  and 
from  this  again  by  the  addition  of  eosin  he  prepares  "Azur  II. -eosin." 
The  latest  formula  for  the  finished  stain  is  as  follows  :  Azur  II. -eosin, 
3gr. ;  Azur  II.,  8  gr. ;  glycerin  (Merck,  chemically  pure),  250  gr. ;  methyl 
alcohol  (Kahll.aum,  I.),  250  gr.  This  stain  has  been  extensively  used 
for  demonstrating  the  spirochsete  pallida,  but  it  can  be  used  for  any 
other  purpose  to  which  the  Romanowsky  stains  are  applicable.  For  the 
spirochiete  the  following  are  Giemsa's  directions  : — 

(1)  Fix  films  in  absolute  alcohol  for  fifteen  to  twenty  minutes,  dry 
with  filter-paper.  (2)  Dilute  stain  with  distilled  water — one  drop  of 
stain  to  1  c.c.  water  (the  mixture  being  well  shaken).  (Sometimes  the 
water  is  made  alkaline  by  the  addition  of  one  drop  of  1  per  cent,  potassium 
carbonate  to  10  c.c.  water.)  (3)  Stain  for  fifteen  minutes.  (4)  Wash  in 
brisk  stream  of  distilled  water.  (5)  Drain  with  filter-paper,  dry,  and 
mount  in  Canada  balsam. 

With  regard  to  the  Jenner  and  Giemsa  stains  it  is  best  to  obtain  the 
solutions  from  Griibler  ready  for  use  ;  the  powder  for  Leishman's  stain 
may  be  obtained  from  the  same  source,  and  the  solution  made  up  by 
the  worker  himself.  Cabot  states  that  Wright's  stain  can  be  obtained 
from  the  Harvard  Co-operative  Society,  Boylston  Street,  Boston, 
U.S.A. 

Neisser's  Stain.—  (a)  The  following  is  the  original  method  introduced 
by  Neisser  as  an  aid  to  the  diagnosis  of  the  diphtheria  bacillus.  Two 
solutions  are  used  as  follows  :  (a)  I  grm.  methylene-blue  (Griibler)  is 
dissolved  in  20  c.c.  of  96  per  cent,  alcohol,  and  to  the  solution  are  added 
950  c.c.  of  distilled  water  and  50  c.c.  of  glacial  acetic  acid  ;  (ft)  2  grins. 
Bismarck-brown  (vesuvin)  dissolved  in  a  litre  of  distilled  water.  Films 
are  stained  in  (a)  for  1-3  seconds  or  a  little  longer,  washed  in  water, 
stained  for  3-5  seconds  in  (6),  dried,  and  mounted.  The  protoplasm  of 
the  diphtheria  bacillus  is  stained  a  faint  brown  colour,  the  granules  a  blue 
colour.  Neisser  considers  that  this  reaction  is  characteristic  of  the 
organism,  provided  that  culture..*  on  Loffler's  serum  are  used  and  examined 
after  9-24  hours'  incubation  at  34°  to  35°  C.  Satisfactory  results  are  not 
always  obtained  in  the  case  of  films  pn-pan-d  from  membrane,  etc.,  but 
there  is  no  doubt  that  here  also  the  method  is  one  of  considerable 
value. 


116  MICROSCOPIC  METHODS 

(b)  The  following  is  Neisser's  modified  cresoidin  method  : — 

1.  Stain  films  for  a  few  seconds  in  a  mixture  of  solutions  A  and  B, 

two  parts  of  the  former  to  one  of  the  latter. 

A.  Methylene-blue      .  .  .  .           1  part. 
Absolute  alcohol    .  '.  .  .         50  parts. 
Glacial  acetic  acid .  ..  '".  .         50      ,, 
Distilled  water      .;  .   .  .  .  1000     „ 

B.  Crystal-violet  (Hochst)  .         ;        ,•          1  part. 
Absolute  alcohol    .  .         .         10  parts. 
Distilled  water     -,         .         ..        .       300     ,, 

2.  Wash  in  water,  and 

3.  Stain  in  cresoidin  solution  (1  :  300)  for  a  few  seconds  (the  cresoidin 

should  be  dissolved   in   warm   water  and  the  solution    then 
filtered). 

4.  Wash  in  water,  dry,  and  mount. 

Sabouraud's  Method  for  Staining  Trichophyta. — Remove  the  fat  from 
the  hair  or  epithelial  squames  witli  chloroform.  Place  in  a  test-tube 
with  10  per  cent,  formol,  and  warm  for  two  or  three  minutes  till  ebullition 
commences.  Wash  well  in  distilled  water,  and  stain  for  one  minute  in 
Sahli's  blue,  which  is  made  up  as  follows  : — 

Distilled  water     .         .         .         .         .         .40  parts. 

'    Saturated  watery  methylene  blue  .         .         .     24     ,, 
5  per  cent,  solution  of  borax  in  water    .  16     ,, 

Mix  the  constituents.  Allow  to  stand  for  a  day,  and  filter.  After 
staining,  wash  in  water,  dehydrate  with  absolute  alcohol,  clear  in  xylol, 
and  mount  in  balsam. 


CHAPTEK  IV. 

METHODS    OF    EXAMINING    THE    PROPERTIES'    OF 
SERUM— PREPARATION     OF      VACCINES  - 
GENERAL    BACTERIOLOGICAL    DIAGNOSIS— IN- 
OCULATION OF  ANIMALS. 

THE  TESTING  OF  AGGLUTINATIVE  AND  SEDIMENTING 
PROPERTIES  OF  SERUM. 

Wright's  Method  of  measuring  Small  Amounts  of  Fluids.— 
It  is  convenient  here  to  describe  this  method.  In  ordinary  work 
fine  calibrated  pipettes  may  be  used  for  measuring  small 
quantities  of  fluids,  but  such  pipettes  are  not  always  available, 
and  by  Wright's  technique  if  a  Gower's  5  c.mm.  haemocytometer 
pipette  be  at  hand  any  measurements  may  be  undertaken — in 
fact,  once  the  pipette  now  to  be  described  (see  Fig.  43)  is  made 
we  are  independent  of  other  means  of  measurement.  A  piece 
of  quill  tubing  is  drawn  out  to  capillary  dimensions,  and  the 
extreme  tip  of  it  is  heated  in  a  peep  name  and  then  drawn  out 
till  it  is  of  the  thickness  of  a  hair,  though  still  possessing  a  bore. 
If  the  point  be  broken  off  this  hair,  and  mercury  be  run  into  the 
tube,  the  metal  will  be  caught  where  the  tube  narrows  and  will 
pass  no  further — in  fact,  though  air  will  pass,  mercury  will  not. 
Into  the  wide  end  of  this  tube  5  c.mm.  of  mercury,  measured 
from  a  Gower's  pipette,  is  run  down  till  it  will  go  no  further. 
A  mark  is  made  on  the  tube  at  the  proximal  end  of  the  mercury, 
which  is  now  allowed  to  run  out,  and  the  tube  is  carefully  cut 
through  at  the  mark.  A  piece  of  ordinary  quill  tubing  is  drawn 
out  and  broken  off  just  below  where  its  narrowing  has  begun, 
the  hair  end  of  the  capillary  tube  is  slipped  through  the  broken- 
off  end,  and  the  tube  is  fixed  in  position  with  wax  as  shown  in 
the  figure.  A  rubber  nipple  placed  on  the  end  of  the  pipette 
completes  the  apparatus.  If  by  pressing  the  nipple  the  air  be 
expelled  from  the  pipette,  and  the  end  dipped  under  mercury, 
exactly  5  c.mm.  will  be  taken  up.  Thus,  when  pressure  on  the 

117 


118 


METHODS  OF  EXAMINING  SERUM 


B 


nipple  is  relaxed,  other  tubes  can  be  very  readily  calibrated  by 
the  mercury  being  expelled  into  them,  and  its  limits  marked  on 
their  bores. 

For  measuring  equal  parts  of  different  fluids,  the  pipette 
shown  in  Fig.  44,  d,  in  connection  with 
agglutination  is  very  useful. 

Methods  of  testing  for  Simple  Ag- 
glutination.— By  agglutination  is  meant 
the  aggregation  into  clumps  of  uniformly 
disposed  bacteria  in  a  fluid ;  by  sedimenta- 
tion the  formation  of  a  deposit  composed 
of  such  clumps  when  the  fluid  is  allowed 
to  stand.  Sedimentation  is  thus  the 
naked-eye  evidence  of  agglutination.  The 
blood  serum  may  acquire  this  clumping 
power  towards  a  particular  organism  under 
certain  conditions ;  these  being  chiefly  met 
with  when  the  individual  is  suffering  from 
the  disease  produced  by  the  organism,  or 
has  recovered  from  it,  or  when  a  certain 
degree  of  immunity  has  been  produced 
artificially  by  injections  of  the  organism. 
The  nature  of  this  property  will  be  dis- 
cussed later.  Here  we  shall  only  give  the 
technique  by  which  the  presence  or  absence 

of  the  p.r°perty  may be  tested-  There  are 

Casing  of  quill  tubing;  two  chief  methods,  a  microscopic  and  a 
B,  rubber  nipple ;  c,  naked-eye,  corresponding  to  the  effects 
Hilary0 *Ube  o°f  F5  mentioned  above.  In  both,  the  essential 
c.mm.  capacity ;  D  to  process  is  the  bringing  of  the  diluted 
E,  hair  capillary.  serum  into  contact  with  the  bacteria 

uniformly  disposed    in    a    fluid.      In    the 

former  this  is  done  on  a  glass  slide,  and  the  result  is  watched 
under  the  microscope ;  the  occurrence  of  the  phenomenon  is 
shown  by  the  aggregation  of  the  bacteria  into  clumps,  and  if  the 
organism  is  motile  this  change '  is  preceded  or  accompanied  by 
more  or  less  complete  loss  of  motility.  In  the  latter  method 
the  mixture  is  placed  in  an  upright  thin  glass  tube ;  sedimenta- 
tion is  shown  by  the  formation  within  a  given  time  (say  from  two 
hours  at  37°  C.  to  twenty-four  hours  at  room  temperature)  of  a 
somewhat  flocculent  layer  at  the  bottom,  the  fluid  above  being 
clear.  Two  points  should  be  attended  to  :  (a)  controls  should 
always  be  made  with  normal  serum,  and  (6)  the  serum  to  be 
tested  should  never  be  brought  in  the  undiluted  condition  into 


METHODS  OF  TESTING  FOR  AGGLUTINATION     119 


contact   with 
following  : — 


the  bacteria.      The  stages  of  procedure  are  the 


- 


1.  Blood  is  conveniently  obtained  by  pricking  the  lobe  of  the   ear, 
which   should   previously  have  been  washed  with  a  mixture  of  alcohol 
and  ether,  and  allowed  to 

dry.  The  blood  is  drawn  /^ 
up  into  a  Wright's  blood- 
capsule  (Fig.  45)  or  into 
the  bulbous  portion  of  a 
<-apillary  pipette,  such  as 
in  Fig.  44,  a.  (These  pip- 
ettes can  be  readily  made 
by  drawing  out  quill  glass- 
tubing  in  a  flame.  It  is 
convenient  always  to  have 
several  ready  for  use.) 
The  pipette  is  kept  in  the 
upright  position,  one  end 
liein^  closed.  For  purposes 
of  transit,  break  off  the 
bulb  at  the  constriction 
and  seal  the  ends.  After 
the  .  serum  has  separated 
from  the  coagulum  the 
bulb  is  broken  through 
near  its  upper  end,  and  tlie 
serum  removed  by  means 
of  another  capillary  pip- 
ette. The  serum  is  then 
to  be  diluted. 

2.  The    serum   may   be 
diluted  (a)  by  means  of  a 
graduated    pipette — either 
a    leucocytorneter    pipette 
(Fig.  44,  6)  or  some  cor- 
responding form.     In  this 
way    successive    dilutions 
of   1  : 10,     1  :  20,    1  :  100, 
etc.  can  be  rapidly  made. 
This  is  the  best  method. 
(b)  By  means  of  a  capillary 
pipette  with  a  mark  on  the 
tube,  the  serum  is  drawn 
up  to  the  mark  and  then 
blown    out    into    a    glass 
capsule  ;   equal  quantities 
of  bouillon  are  successively 

measured  in  the  same  way,  and  added  till  the  requisite  dilution  is 
obtained,  (c)  By  means  of  a  platinum  needle  with  a  loop  at  the  end 
(Delepine's  method).  A  loopful  of  serum  is  placed  on  a  slide,  and  the 
dfsiwl  number  of  similar  loopfuls  of  bouillon  are  separately  placed 
around  on  the  slide.  The  drops  are  then  mixed. 

A  very  convenient  and  rapid  method  of  combining  the  steps  1  and  2 


FIG.  44. — Tubes  used  in  testing  agglutinating 
and  sediinenting  properties  of  serum. 


120  METHODS  OF  EXAMINING  SERUM 

is  to  draw  a  drop  of  blood  up  to  the  mark  1  or  '5  on  a  leucocytometer 
pipette,  and  draw  the  bouillon  after  it  till  the  bulb  is  filled.  A  dilution 
of  10  or  20  times  is  thus  obtained.  Then  blow  the  mixture  into  a 
U-shaped  tube  (Fig.  44,  c),  and  ceutrifugalise  or  simply  allow  the  red 
corpuscles  to  separate  by  standing.  (In  this  method,  of  course,  the 
dilution  is  really  greater  than  if  pure  serum  were  used,  and  allowance 
must  therefore  be  made  in  comparing  results.)  The  presence  of  red 
corpuscles  is  no  drawback  in  the  case  of  the  microscopic  method,  but  when 
sedimentation  tubes  are  used  the  corpuscles  should  be  separated  first. 

3.  The  bacteria  to  be  tested  should  be  taken  from  young  cultures, 
preferably  not  more  than  twenty-four  hours   old,  incubated  at  37°  C. 
They  may  be  used  either  as  a  bouillon  culture  or  as  an  emulsion  made 
by  adding  a  small  portion  of  an  agar  culture  to  bouillon  or  '8  per  cent, 
solution  of  sodium  chloride.     In  tlie  latter  case  the  mass  of  bacteria  on  a 
platinum  loop  should  be  gently  broken  down  at  the  margin  of  the  fluid  in 
a  watch-glass.     When  a  thick  turbidity  is  thus  obtained,  any  remaining 
fragments  should  first  be  removed,  and  then  the  organisms  should  be  uni- 
formly mixed  with  the  rest  of  the  fluid.     The  bacterial  emulsion  ought  to 
have  a  faint  but  distinct  turbidity.    (When  the  exact  degree  of  sedimenting 
power  of  a  serum  is  to  be  tested — expressed  as  the  highest  dilution  in  which 
it  produces  complete  sedimentation  within  twenty-tour  hours — a  standard 
quantity  (by  weight)  of  bacteria  must  be  added  to  a  given  quantity  of 
bouillon.     This  is  not  necessary  for  clinical  diagnosis. ) 

4.  To   test    microscopically,    mix    equal    quantities    (measured   by   a 
marked    capillary   pipette)    of    the    diluted    serum    and    the    bacterial 
emulsion  on  a  glass  slide,  cover  with  a  cover-glass,  and  examine  under 
the  microscope.     The  form  of  glass  slide   used  for  hang-drop  cultures 
(Fig.    27)   will   be   found  very  suitable.     The  ultimate  dilution  of  the 
serum  will,  of  course,  be  double  the  original  dilution. 

To  observe  sedimentation,  mix  equal  parts  of  diluted  serum  and  of 
bacterial  emulsion,  and  place  in  a  thin  glass  tube — a  simple  tube  with 
closed  end  or  a  U-tube.  Keep  in  upright  position  for  twenty-four 
hours.  One  of  Wright's  sedimentation  tubes  is  shown  in  Fig.  44,  d. 
Diluted  serum  is  drawn  up  to  fill  the  space  mn,  a  small  quantity  of  air 
is  sucked  up  after  it  to  separate  it  from  the  bacterial  emulsion,  which 
is  then  drawn  up  in  the  same  quantity  ;  the  diluted  serum  will  then 
occupy  the  position  Id.  The  fluids  are  then  drawn  several  times  up 
into  the  bulb,  and  returned  to  the  capillary  tube  so  as  to  mix,  and  finally 
blown  carefully  down  close  to  the  lower  end,  which  is  then  sealed  off. 
The  sediment  collects  at  the  lower  extremity. 

It  is  often  important  to  observe  not  merely  the  fact  that  agglutination 
occurs,  but  also  the  weakest  concentration  of  the  serum  with  which  the 
reaction  can  be  obtained. 

Measurement  of  Group  Agglutinins. — In  the  case  of  certain 
groups  of  allied  organisms, — notably  the  b.  coli  and  its  allies,— 
it  has  been  found  that  when  a  serum  clumps  one  member  of  the 
group  it  frequently  also  clumps  the  allied  forms.  If  the  greatest 
dilution  with  which  agglutination  is  obtained  be  estimated,  the 
end-points  for  the  different  strains  affected  are  usually  found  to 
differ.  The  determination  of  the  end-point  is  important,  as  the 
disease  condition  from  which  the  serum  is  derived  is  generally 
caused  by  the  organism  which  is  clumped  in  highest  dilution. 


METHOD  OF  TESTING  AGGLUTININS  121 

Tn  comparing  the  effect  of  a  serum  on  different  bacteria,  the 
sedimentation  method  is  usually  employed.  A  series  of 
emulsions  of  the  clitic- rent  bacteria  to  be  tested  is  prepared  by 
scraping  off  the  growth  on  an  agar  tube,  and  suspending  in 
bouillon.  Each  of  these  should  approximately  contain  the  same 
number  of  bacteria  per  unit  volume.  This  is  attained  by  using 
emulsions  of  equal  opacity,  as  judged  of  by  noting  the  point  at 
which  transparency  to  some  arbitrary  standard  such  as  a 
particular  type  or  set  of  parallel  lines  ceases.  A  given  amount 
of  each  emulsion  is  now  mixed  with  different  dilutions  of  the 
serum  to  be  tested,  the  mixtures  are  all  made  up  to  the  same 
volume,  say  1  c.c.,  and  the  tubes  placed  at  37°  C.  for  two  or 
three  hours.  The  results  are  then  read,  the  tubes  are  set  aside 
at  room  temperature  for  twenty-four  hours,  and  read  again; 
usually  the  two  readings  correspond. 

The  Absorption  Method  of  testing  Agglutinins. — This 
method  is  applied  under  circumstances  similar  to  those  of  the 
last,  namely,  when  several  agglutinins  acting  on  allied  organisms 
are  present  in  a  serum.  The  principle  is  to  remove  all  the 
agglutinins  acting  on  one  organism,  and  to  study  the  properties 
of  those  which  remain.  In  practice  the  method  consists  in 
adding  to  the  serum  a  mass  of  one  of  the  bacteria  of  the  group 
under  study  (the  organisms  being  scraped  off  an  agar  slope), 
allowing  the  mixture  to  stand  at  37°  C.  for  two  or  three  hours, 
and  then  separating  the  bacteria  with  the  centrifuge.  The 
supernatant  clear  fluid  is  now  pipetted  off,  and  its  agglutinating 
pro] Arties  studied  on  the  other  members  of  the  bacterial  group 
either  by  sedimentation  or  by  the  microscopic  method.  The 
use  of  the  method  is  to  aid  in  differentiating  which  member  of 
a  bacterial  group  is  causally  related  to  the  condition  from  which 
the  serum  is  obtained,  and  an  example  of  its  application  for  this 
purpose  will  be  found  in  the  chapter  on  typhoid  fever  (p.  375). 
It  has  also  been  used  by  Park  and  Collins  and  by  Bainbridge 
for  identifying  strains  of  organisms  of  the  typhoid-coli  group. 
Here  the  principle  is  that,  when  an  unknown  strain  belonging 
to  such  a  bacterial  group  is  under  investigation,  if  its  capacities 
for  absorbing  agglutinins  from  a  serum  containing  a  mixture 
of  such  are  the  same  as  those  of  an  already  recognised  strain, 
then  the  two  are  probably  identical. 

OPSONIC  METHODS. 

Method  of  measuring  the  Phagocytic  Capacity  of  the 
Leucocytes. — This  was  first  done  by  Leishmau  by  a  very 


122  METHODS  OF  EXAMINING  SERUM 

simple  method,  as  follows  :  A  piece  of  quill  tubing  is  drawn  out 
to  a  capillary  diameter  so  as  to  make  a  pipette  about  6  inches 
long.  The  point  is  broken  off,  and  a  rubber  nipple  adjusted  to 
the  wide  end ;  a  mark  is  made  with  an  oil  pencil  about  three- 
quarters  of  an  inch  above  the  orifice.  Blood  is  drawn  from  the 
finger  up  to  the  mark,  then  an  air-bubble  is  allowed  to  pass  in. 
A  thin  emulsion  of  the  bacterium  to  be  tested  having  been  pre- 
pared, a  quantity  of  this  is  also  drawn  up  to  the  mark.  The 
two  fluids  are  thoroughly  mixed  by  being  first  blown  out  on 
to  a  sterile  slide  and  then  being  drawn  back  into  the  pipette 
and  expelled, — this  being  repeated  several  times.  A  cover-glass 
is  placed  over  the  drop,  and  the  slide  is  placed  in  the  incubator 
at  37°  C.  for  fifteen  minutes.  The  cover-glass  is  then  slipped 
off  so  as  to  make  a  film  preparation,  which  in  the  case  of 
ordinary  bacteria  may  be  stained  by  Leishman's  method.  The 
number  of  bacteria  present  in,  say,  fifty  polymorphonuclear  cells 
successively  examined  is  determined,  and  an  average  struck.  The 
method  was  first  used  for  showing  that  in  cases  of  staphylococcus 
infection  the  average  number  of  bacteria  taken  up  was  less  than 
in  a  control  in  which  the  same  bacterial  emulsion  was  exposed 
to  the  blood  of  a  healthy  individual.  In  making  such  an 
observation,  drops  from  the  two  mixtures  are  placed  on  the  same 
slide  under  separate  cover-glasses,  and  the  preparation  incubated. 
One  cover  is  then  slipped  to  one  end  of  the  slide,  and  the  other 
to  the  other, — the  two  films  being  then  stained  as  one. 

Leishman's  method  gives  what  may  be  called  the  total  phago- 
cytic  capacity  of  the  blood,  but  according  to  Wright's  view  the 
process  of  phagocytosis  in  blood  outside  the  body  is  not  a 
simple  one,  and  before  a  leucocyte  takes  up  a'  bacterium  the 
latter  must  be  acted  on  in  some  way  by  substances  present  in 
the  serum,  which  Wright  calls  opsonins  (see  Immunity).  The 
technique  by  which  the  actions  of  these  opsonins  is  studied 
has  been  elaborated  by  Wright  and  his  co-workers  in  connec- 
tion with  bacterial  vaccines,  especially  in  relation  to  infection 
by  the  pyogenic  cocci  and  the  tubercle  bacillus.  This  technique 
involves  (1)  the  preparation  of  the  bacterial  emulsion,  (2)  the  pre- 
paration of  the  leucocytes,  (3)  the  preparation  of  samples  of  (a) 
serum  from  a  normal  person,  and  (b)  serum  from  the  infected 
person. 

(1)  Preparation  of  Bacterial  Emulsion. — In  the  case  of  the 
pyogenic  cocci,  a  little  of  a  twenty-four  hour  living  culture  off 
a  sloped  agar  tube  is  taken  and  rubbed  up  in  a  watch-glass  with 
•85  per  cent,  saline.  The  mixture  is  placed  in  a  tube  and  centri- 
fugalised,  so  as  to  deposit  any  masses  of  bacteria  which  may  be 


PREPARATION  OF  LEUCOCYTES      123 

present.  Only  by  experience  can  a  knowledge  be  gained  of 
the  amount  of  culture  to  be  used  in  the  first  instance,  but  the 
resultant  emulsion  usually  should  exhibit  only  the  merest  trace 
of  cloudiness  to  the  naked  eye.  Wright  states  it  will  then  con- 
tain from  7000  to  10,000  million  bacteria  per  c.cm.  If  too 
strong  an  emulsion  be  used,  the  leucocytes  may  take  up  so  many 
organisms  that  these  cannot  be  accurately  enumerated.  In  the 
case  of  the  tubercle  bacillus,  as  short  a  variety  of  the  organism 
as  possible  should  be  selected,  and  a  mass  of  growth  off  a  solid 
medium  is  taken  (bacilli  in  mass  can  be  obtained  in  the  market 
from  wholesale  chemists)  and  is  well  washed  with  changes  of 
distilled  water,  drained  on  filter  paper  in  a  Petri  dish,  and 
thoroughly  rubbed  up  with  a  little  1'5  per  cent,  saline  in  an 
agate  mortar,  so  as  to  disintegrate  the  bacterial  masses  and  get 
an  emulsion  composed  as  far  as  possible  of  individual  bacilli.  It 
is  extremely  difficult  to  obtain  a  good  emulsion  of  tubercle  bacilli, 
i.e.  one  that  shall  consist  as  far  as  possible  of  separate  bacilli 
— on  the  one  hand  without  clumps,  and  on  the  other  without 
portions  of  disintegrated  bacilli.  The  rubbing-up  in  the  mortar, 
which  usually  occupies  many  hours,  must  be  done  very  slowly 
and  gently  with  a  very  light  pestle,  and  the  manipulation  must 
be  frequently  controlled  by  microscopic  observation.  A  thick 
cream  should  be  obtained,  and  this  should  be  sterilised  by 
steaming  for  half  an  hour  on  three  successive  days.  Before 
sterilisation  it  is  convenient  to  seal  up  the  stock  emulsion  in 
small  quantities  in  a  number  of  pieces  of  quill  tubing,  so  that 
in  the  subsequent  procedures  only  small  portions  of  the  emulsion 
are  exposed  to  aerial  contamination  at  one  time.  For  actual 
use,  one  of  those  tubes  is  opened,  a  little  is  withdrawn  with  a 
sterile  pipette,  and  a  weak  emulsion  made  in  the  same  way  as 
with  the  staphylococcus,  except  that  1  '5  per  cent,  saline  is  used. 
The  stock  tube  may  be  sealed  with  wax  and  kept  for  use  again. 
A  fresh  emulsion  ought  to  be  made  up  for  each  day's  work. 

(2)  Preparation  of  Leucocytes. — Here  the  observer  uses  his 
own  blood  cells.  A  1*5  per  cent,  solution  of  sodium  citrate  in 
•85  per  cent,  sodium  chloride  is  prepared.  This  is  placed  in  a 
glass  tube  3  inches  long,  made  by  drawing  out  a  piece  of 
half-inch  tubing  to  a  point,  the  tube  being  filled  nearly  to  the 
brim.  A  handkerchief  being  bound  round  the.  finger,  this  is 
now  pricked,  and  the  blood  allowed  to  flow  directly  into  the 
fluid,  to  the  bottom  of  which  it  sinks.  The  tube  ought  to  be 
inverted  between  the  addition  of  every  few  drops  of  blood,  so  as 
to  bringc'the  blood  in  contact  with  the  citrate  and^'prevent 
coagulation.  The  equivalent  of  about  ten  to  twenty  drops  of 


124 


METHODS  OF  EXAMINING  SERUM 


blood  should  be  obtained.  The  diluted  blood  is  then  centri- 
f ugalised,  and  when  the  corpuscles  are  separated  the  supernatant 
fluid  is  removed,  '85  per  cent,  saline  is  substituted,  and  the 
centrifugalisation  repeated.  The  fluid  is  again  removed,  care 
being  taken  not  to  disturb  the  layer  of  white  cells  lying  on  the 
top  of  the  red  corpuscles.  This  layer  is  then  pipetted  off  into 
a  watch-glass  or  tube,  and  the  leucocytes  required  are  thus 
obtained. 

(3)  Preparation  of  the' Sera. — The  serum  whose  sensitising 
effect  on  the  bacteria  it  is  desired  to  test  is  obtained  by  Wright 
as  follows  :  A  "  blood-capsule  "  is  made  by  drawing  a  piece  of 
No.  3  quill  tubing  into  the  shape  shown  in  Fig.  45,  the  part  not 
drawn  out  being  about  1  inch  in  length.  It  is  convenient  to 
make  a  number  of  these  capsules  at 
one  time,  and  to  draw  off  their 
extremities  and  seal  them  in  the 
flame.  For  use,  the  tips  of  both 
extremities  are  broken  off,  the  finger 
is  pricked,  and  blood  allowed  to  pass 
into  the  capsule  through  the  bent 
limb  till  the  capsule  is  about  half 
full.  The  air  remaining  in  the 
capsule  is  rarefied  by  passing  the 
straight  end  through  a  flame  and 
then  sealing  it  off.  By  this  manipul- 

FIG.  45. -Wright's  blood-cap-  ation  the  blood  is  sucked  over  the 
sule,  and  method  of  filling  bend  into  the  straight  part  of  the 
same,  tube,  and  the  bent  end  is  now  also 

sealed  off  or  closed  with  wax.     It  is 

well  to  shake  the  blood  down  towards  the  closed  straight  end, 
care  being  taken  to  previously  allow  the  glass  to  cool  sufficiently. 
The  capsule  is  now  hung  by  the  bend  on  the  edge  of  a  centri- 
fuge tube,  and  the  serum  separated  by  spinning  the  instrument. 
In  any  particular  case  a  capsule  of  serum  from  the  infected 
person  and  one  from  a  normal  individual  are  prepared. 

The  emulsion,  corpuscles,  and  serum  being  thus  prepared, 
the  next  step  is  to  mix  them.  This  is  done  by  taking  a  piece 
of  quill  tubing  and  drawing  it  out  to  a  capillary  point  so  as  to 
make  a  pipette  about  8  inches  long;  on  the  thick  end  of 
this  a  rubber  teat  is  fixed,  and  about  1  inch  from  the  capillary 
point  a  mark  is  made  with  an  oil  pencil.  From  the  watch-glass 
containing  the  separated  leucocytes  a  portion  is  sucked  up  to 
the  mark,  and  then  an  air-bubble  is  allowed  to  pass  in.  A 
similar  portion  of  the  serum  is  drawn  up,  and  then  another 


PREPARATION  OF  THE  SERA  125 

air-bubble,  and  finally  a  similar  portion  of  the  bacterial 
emulsion.  The  three  droplets  are  carefully  blown  on  to  a  slide, 
and  are  thoroughly  mixed  with  one  another  by  being  alternately 
drawn  up  into  the  tube  and  expelled  ten  times.  The  mixture 
is  then  drawn  into  the  tube,  and  the  end  sealed  off  in  the  flame. 
The  rubber  nipple  is  removed,  and  the  tube  placed  in  the 
incubator  at  37°  for  fifteen  minutes.  A  slide  is  now  prepared 
by  rubbing  it  once  or  twice  with  very  fine  emery  paper  (No.  000) 
and  thoroughly  wiping  it.  This  is  a  procedure  adopted  by 
Wright  to  cause  an  evenly  distributed  film  to  be  made.  The 
tube  being  removed  from  the  incubator  and  the  end  broken  off, 
its  contents  are  again  mixed  by  expelling  and  drawing  up  into 
the  tube.  A  minute  droplet  is  placed  on  the  prepared  slide, 
and  by  means  of  the  edge  of  the  end  of  another  slide  a  film 
is  made,  which  is  then  dried  and  is  ready  for  staining.  The 
spreader  should  be  slightly  narrower  than  the  slide  on  which 
the  film  is  made ;  in  this  way  the  film  has  two  definite  edges — 
a  fact  of  importance,  as  the  leucocytes  are  usually  in  greatest 
abundance  near  these  edges.  Films  containing  staphylococci 
are  stained  either  by  Leishman's  stain  (</.v.)  or  with  carbol- 
thionin  blue.  In  the  former  case  no  fixation  is  necessary,  in 
the  latter  it  i.s  usual  to  fix  in  saturated  perchloride  of  mercury 
for  one  and  a  half  minutes,  wash  in  water,  and  then  stain.  With 
tubercle  films  the  following  is  the  procedure  :  The  film  is  fixed  for 
twoininutfs  in  |  »erehloride  of  mercury,  washed  thoroughly,  stained 
with  carbol-fuchsin  as  usual,  decolorised  with  2*5  per  cent, 
sulphuric  acid,  cleared  with  4  per  cent,  acetic  acid,  counter- 
stained  with  watery  solution  of  methylene-blue,  and  dried. 

In  applying  the  technique  two  preparations  are  made,  in  both 
of  which  the  same  emulsion  and  the  same  leucocytes  are  em- 
ployed ;  but  in  one  the  bacteria  have  been  exposed  to  the  serum 
of  the  infected  individual  under  observation,  and  in  the  other 
to  that  of  a  normal  person, — usually  the  observer  himself, — or 
better  still,  to  a  mixture  of  sera  from  several  normal  persons. 
Each  of  these  preparations  is  now  examined  microscopically  with 
a  movable  stage,  the  number  of  bacteria  in  the  protoplasm  of  at 
least  fifty  polymorphonucleated  leucocytes  is  counted,  and  an 
average  per  leucocyte  struck  (this  is  often  called  the  "  phagocytic 
index  ") ;  the  proportion  which  this  average  in  the  case  of  the 
abnormal  serum  bears  to  the  average  in  the  preparation  in  which 
the  healthy  serum  was  used  constitutes  the  opsonic  index — that 
of  healthy  serum  being  reckoned  as  unity. 

The  reliability  of  the  opsonic  method,  of  course,  depends  on 
whether  or  not  the  phagocytic  activity  of  the  cells  counted 


126  METHODS  OF  EXAMINING  SERUM 

represents  the  phagocytic  activity  of  the  cells  in  the  preparation. 
Considerable  controversy  has  arisen  on  this  point.  The  general 
result  may  be  said  to  be  that  where  such  organisms  as  the 
pyogenic  cocci  are  concerned,  the  ordinary  opsonic  technique 
gives  on  the  whole  reliable  results.  In  the  case  of  the  tubercle 
bacillus,  there  is  considerable  difference  of  opinion.  Generally 
speaking,  it  may  be  said  that  indices  varying  between  '8  and 
1  *2  are  to  be  reckoned  as  unity — that  is  to  say,  that  no  deduction 
can  be  drawn  from  indices  falling  between  these  limits.  In  the 
case  of  such  organisms  as  those  of  the  coli-typhoid  group  a.nd 
cholera,  which  are  susceptible  to  bacteriolytic  influences  in  the 
serum,  it  may  be  necessary  to  heat  the  sera  of  the  patient  and 
observer  for  half  an  hour  at  55°  C.  This  destroys  any  com- 
plement present  and  prevents  bacteriolysis  occurring.  In  the 
case  of  the  b.  typhosus  the  virulence  of  the  strain  employed  has 
been  shown  to  be  an  important  factor. 

Several  modifications  of  Wright's  technique  have  been 
suggested.  Thus  Klien,  instead  of  enumerating  the  bacteria 
ingested,  takes  a  series  of  dilutions  of  the  serum  and  estimates 
the  dilution  with  which  capacity  for  opsonising  bacteria  dis- 
appears (or  at  any  rate  the  dilution  with  which  the  phagocytic 
index  falls  below  '5).  The  content  of  the  patient's  serum  and 
of  that  of  the  observer  may  be  thus  compared,  or  the  course  of 
an  immunisation  may  be  followed  by  making  daily  observations 
of  the  content  in  opsonin.  In  another  modification  of  Wright's 
technique  Simon  compares  not  the  numbers  of  bacteria  ingested, 
but  the  percentages  of  cells  containing  bacteria  to  those  not 
containing  bacteria.  This  he  calls  the  "  percentage  index,"  and 
he  states  that  the  figure  thus  obtained  corresponds  very  closely 
to  the  ordinary  opsonic  index;  he  claims  that  the  method 
eliminates  some  of  the  errors  which  may  arise  in  the  use  of  the 
ordinary  technique  if  only  a  relatively  small  number  of  phago- 
cyting  cells,  such  as  50,  be  examined. 

BACTERICIDAL  METHODS— DEVIATION  OF  COMPLEMENT. 

The  Estimation  of  the  Bactericidal  Action  of  Serum. — This 
may  be  carried  out  by  various  methods,  of  which  those  of 
Neisser  and  Wechsberg  and  of  Wright  may  be  given  as  examples. 
In  the  former,  the  effects  of  varying  amounts  of  serum  on  the 
same  amounts  of  bacteria  are  observed  by  means  of  plate 
cultures ;  in  the  latter,  the  number  of  bacteria  which  can  be  com- 
pletely killed  off  by  a  given  quantity  of  serum  is  ascertained. 
In  carrying  out  experiments  of  this  kind  it  is  convenient  to  have 


BACTERICIDAL  METHODS  127 

a  number  of  small  test-tubes  sterilised  and  plugged  with  cotton- 
wool. We  can  then  make  any  required  dilution  of  a  young 
bacterial  culture  in  bouillon  as  follows :  To  each  of  a  number 
of  tubes  we  add  '9  c.c.  of  '8  per  cent,  solution  of  sodium  chloride. 
To  the  first  tube  (a)  we  add  '1  c.c.  of  the  bacterial  culture,  and 
thoroughly  shake  up  the  mixture ;  to  the  second  (6)  we  add 
•1  c.c.  of  the  contents  of  (a),  and  shake  up ;  to  the  third  tube 
(c)  we  add  '1  c.c.  of  the  contents  of  (6),  and  so  on.  It  is  thus 
evident  that  '1  c.c.  of  the  contents  of  (a)  will  correspond  to 
'01  c.c.,  and  '1  c.c.  of  (b)  to  '001  c.c.  of  the  original  culture  ;  any 
minimi  fraction  can  thus  be  readily  obtained.  In  the  making 
of  all  mixtures  of  serum  and  bacteria  it  is  essential  that  none  of 
the  latter  shall  escape  the  action  of  the  former,  e.y.  by  remaining 
on  a  part  of  the  mixing  vessel  with  which  the  serum  does  not 
come  in  contact. 

(a)  Method   of  Neisser  and    Wechsbery. — A  series  of  small 
plugged  sterile  tubes  is   taken,  and  to  each  we  add  '5  c.c.  of 
•8  per  cent,  sodium  chloride  solution,  and  a  given  quantity,  say 
•5^5.  c.c.,  of  a  young  bouillon   culture  to  be  tested.     To  the 
several  tubes  in  series  we  then    add   varying   amounts  of  the 
fresh  serum  whose  action  is  to  be  observed,  e.y.  '2  c.c.,  '1  c.c., 
•05  c.c.,   '025  c.c.,  etc.     The   contents   of  each  tube  are  then 
made  up  to  1  c.c.  with  salt  solution,  and  a  few  drops  of  sterile 
bouillon  are  added  to  each  tube.     The  tubes  are  then  well  shaken 
and   placed  in  the  incubator  at  37°  C.  for  three  hours,  to  allow 
the  serum  to  act.    (Of  course  several  series  of  such  tubes  may  be 
prepared  and  placed  in  the  incubator  for  varying  periods  of  time  ; 
we  can  thus  observe  when   the  bactericidal  effect  reaches  the 
maximum.)     At  the  end  of  the  given  period  of  time  a  small 
quantity,  say  '05  c.c.,  of  the  contents  of  each  tube  is  added  to  a 
tube  of  melted  agar  (cooled  to  about  40°  C.) ;  each  agar  tube  is 
then  shaken,  and  the  contents  are  poured  out  into  a  sterile  Petri 
capsule.     The  other  tubes  are  similarly  treated,  and  the  Petri 
capsules  are  placed  in  the  incubator  for  a  suitable  period  of  time. 
The   number   of   colonies    in    each    can    then    be    noted.      Of 
course  gelatine  can  be  substituted  for  the  agar  in  the  plates  if 
desired. 

(b)  Wright's  Method. — A  twenty-four  hours1  bouillon  culture  is 
used,  and  various  dilutions  with  sterile  bouillon  are  made  according 
to  the  method  described  on  p.   58  :  thus  5-,  10-,  20-,  50-,  100-, 
1000-,  etc.,  fold  dilutions  may  be  prepared.      A  small  quantity, 
.say  1  c.mm.,  of  the  fresh  serum  to  be  tested  is  mixed  with  an 
equal  amount  of  the  bacterial  culture,  and  the  mixture  is  placed 
in  a  small  capillary  tube  which  is  sealed  at  the  ends ;  similar 


128  METHODS  OF  EXAMINING  SERUM 

mixtures  of  equal  parts  of  serum  and  of  each  of  the  dilutions  of 
culture  are  prepared  and  treated  in  the  same  way.  The  tubes 
are  then  placed  in  the  incubator  for  eighteen  to  twenty-four 
hours  at  37°  C.,  and  at  the  end  of  that  time  the  contents  of 
each  are  tested  as  regards  sterility  by  means  of  cultures.  In 
this  way  the  greatest  dilution  in  which  the  bacteria  are  com- 
pletely killed  off  is  ascertained.  The  number  of  bacteria  in 
the  original  culture  per  c.mm.  can  be  counted  by  the  method 
given  on  p.  70,  and  thus  the  total  number  of  bacteria  killed 
off  by  the  quantity  of  serum  used  can  readily  be  calculated. 

As  will  afterwards  (see  chapter  on  Immunity)  be  described  in 
greater  detail,  when  an  animal  is  immunised  against  a  particular 
bacterium  the  bactericidal  action  of  its  serum  may  be  greatly 
increased,  and  this  depends  on  the  development  of  a  particular  sub- 
stance called  an  immune-body,  which  is  comparatively  thermo- 
stable and  is  not  destroyed  at  55°  C.  To  analyse  the  bactericidal 
properties  of  such  a  serum,  it  should  in  the  first  place  be  heated 
in  order  to  destroy  the  normal  complement.  Then  to  each  of  a 
series  of  sterile  tubes  we  add  (a)  a  quantity  of  normal  unheated 
serum  insufficient  of  itself  to  destroy  the  bacteria,  (b)  a  given 
amount  of  the  bacterial  culture,  and  (c)  varying  amounts  of  the 
heated  immune-serum — •].,  '01,  '001,  etc.  c.c.  In  this  way  we 
can  find  the  quantity  of  the  immune-serum  which  gives  the 
maximum  bactericidal  action. 

In  some  cases,  however,  when  an  animal  is  immunised  against 
a  given  bacterium,  or  when  a  patient  is  infected  with  the 
organism,  the  serum  may  not  have  increased  bactericidal  action, 
but  nevertheless  contains  an  immune-body  which  leads  to  the 
absorption  or  fixation  of  complement.  In  other  wrords,  the 
immune-body  is  a  substance  which,  along  with  the  corresponding 
or  homologous  bacterium,  binds  complement  (p.  130).  In  order, 
however,  to  explain  the  methods  by  which  the  fixation  of  com- 
plement may  be  demonstrated,  we  must  first  of  all  give  some 
facts  with  regard  to  hsemolytic  sera. 

Methods  of  Haemolytic  Tests.— A  hamiolytic  serum  is  usually 
prepared  by  injecting  the  red  corpuscles  of  an  animal  into  the 
peritoneum  of  an  animal  of  different  species — the  corpuscles  of 
the  ox  are  most  frequently  used,  and  the  rabbit  is  the  most 
suitable  animal  for  injection.  The  corpuscles  ought  to  be  com- 
pletely freed  of  serum  by  repeatedly  washing  them  in  sterile 
salt  solution,  and  centrifugalising.  An  injection  of  the  corpuscles 
of  5  c.c.  of  ox's  blood,  followed  by  two  injections,  each  of  10  c.c., 
at  intervals  of  eight  days,  will  usually  give  an  active  serum.  The 
animal  should  be  killed  by  bleeding  it,  aseptically  as  far  as  poss- 


METHODS  OF  ELEMOLYTIC  TESTS  129 

ible,  seven  to  ten  days  after  the  last  injection ;  the  serum  which 
separates  may  be  collected  in  suitable  lengths  of  quill  glass- 
tubing  drawn  out  at  the  ends,  which  are  afterwards  sealed  in 
the  flame.  To  ensure  sterility  when  tbe  serum  is  to  be  kept 
some  time,  it  is  advisable  to  heat  it  for  an  hour  at  55°  C.  on 
three  successive  days;  we  have  always  found  that  serum  treated 
in  this  way  remains  sterile.  It  is,  of  course,  devoid  of  comple- 
ment. The  test  amount  of  corpuscles  is  usually  1  c.c.  of  a 
5  per  cent,  suspension  of  corpuscles  in  '8  per  cent,  sodium 
chloride  solution  :  that  is,  the  corpuscles  of  5  c.c.  blood  are 
completely  freed  of  serum  by  repeatedly  washing  in  salt  solution, 
and  then  salt  solution  is  added  to  make  up  100  c.c.  In  any 
investigation  it  is  necessary  to  obtain  the  minimum  hajmolytic 
dose  (M.H.D.)  of  the  immune-body  and  of  the  complement  to 
be  used.  (It  is  to  be  noted  that  as  complement  does  not 
increase  during  immunisation,  the  haemolytic  dose  of  the  fresh 
scrum  will  come  far  short  of  representing  the  amount  of 
immune-body  present.)  In  testing  the  dose  of  immune-body, 
the  fresh  serum  to  be  used  as  complement  must  be  devoid  of 
htemolytic  action  (in  the  present  instance  rabbit's  serum  will  be 
found  suitable),  and  more  than  sufficient  to  produce  lysis  with 
immune-body  is  added  to  each  of  a  series  of  tubes.  Varying 
amounts  of  immune-body  are  added  to  the  tubes,  the  contents 
are  shaken,  made  up  to  1*5  c.c.,  and  incubated  for  two  hours. 
The  amount  of  lysis  is  then  noted,  and  the  tubes  are  placed  in 
a  cool  chamber  till  next  morning,  when  a  final  reading  is  taken. 
The  smallest  amount  of  immune-body  which  gives  complete 
lysis  is,  of  course,  the  M.H.D.  :  sometimes  this  may  be  as  low 
as  '001  c.c.  for  the  test  amount  of  corpuscles.  When  further 
observations  are  to  be  continued  on  the  same  day,  the  reading 
after  incubation  must  be  taken  as  the  working  standard.  To 
estimate  the  M.H.D.  of  complement,  proceed  in  a  corresponding 
manner;  to  each  of  a  series  of  tubes  add  several  doses  of 
immune-body,  and  then  to  the  several  tubes  different  amounts 
of  complement.  The  activity  of  a  serum  as  complement  varies 
considerably,  and  each  sample  must  be  separately  tested.1  The 
above  will  serve  as  an  indication  of  the  fundamental  methods ; 
for  further  details,  special  papers  on  the  subject  must  be 
consulted.  Corpuscles  treated  with  sufficient  immune-body  to 

1  Complement  is  a  substance  which  rapidly  (often  within  twenty-four  hours) 
loses  its  strength  when  kept  at  room  temperature.  It  can,  however,  be  pre- 
•-.•rved  for  a  considerable  time  at  or  near  its  original  strength  if  it  be  kept  frozen. 
Even  if  this  be  done,  however,  the  strength  of  the  complementary  serum  must 
l>e  titred  at  the  commencement  of  every  experiment  in  which  it  is  employed. 


130  METHODS  OF  EXAMINING  SERUM 

produce    complete    lysis    on   the    addition    of    complement    are 
usually  spoken  of  as  sensitised  corpuscles. 

The  Removal  of  Blood-Samples  from  Rabbits,  etc. — In  such  work  as 
that  just  described,  it  is  often  convenient  to  watch  the  progress  of  an 
immunisation  procedure  by  removing  a  sample  of  blood  without  the 
animal  being  killed.  With  proper  care  any  amount  of  blood  up  to  one- 
third  of  that  contained  in  the  body  can  be  removed  from  the  ear  vein  of 
a  rabbit.  The  animal,  which  must  not  be  flurried,  is  placed  on  a  bench, 
and  its  body  kept  warm  by  being  covered  with  a  cloth.  The  root  of  the 
ear  should  be  shaved  over  the  marginal  vein,  the  hairs  on  the  edge  of  the 
ear  should  also  be  clipped  short.  It  is  best  to  have  the  ear  dry,  as  the 
evaporation  of  a  fluid  causes  contraction  of  the  vessels.  In  a  great  deal 
of  hsemolytic  work  absolute  sterility  of  the  sample  is  not  necessary,  so 
that  washing  the  ear  is  not  required.  When  sterile  blood  is  desired,  the 
precautions  detailed  on  p.  44  may  be  applied.  A  frosted  incandescent 
electric  lamp,  such  as  is  used  for  microscopic  illumination,  is  placed  lighted 
an  inch  or  two  from  the  ear.  The  left  hand  of  the  operator  should  cover 
the  animal's  head  in  front  of  the  ears,  the  thumb  and  index  finger  being 
left  free  to  compress  the  vein  at  the  root  of  the  ear.  In  this  way  not 
only  is  the  animal's  eye  protected  from  the  glare  of  the  lamp,  but  the 
distance  of  the  latter  from  the  ear  can  be  regulated  so  as  to  keep  it  at 
what  to  the  operator's  hand  is  a  pleasant  warmth.  In  a  minute  or  two 
the  ear  vessels  will  dilate,  and  the  vein,  being  compressed  at  the  root,  a 
lateral  opening  is  made  with  a  bayonet-pointed  surgical  needle  (the 
triangular-pointed  needles  supplied  with  the  Gowers-Haldane  hsemo- 
globinometer  are  also  very  suitable),  and  the  blood  allowed  to  drop  into  a 
sterile  test-tube.  Usually  waves  of  contraction  of  the  ear  vessels  will  be 
observed  to  occur,  the  passing  off  of  which  must  be  waited  for,  and  from 
time  to  time  the  clot  must  be  gently  squeezed  out  of  the  opening  in  the 
vein  with  the  flat  side  of  the  needle,  or  it  may  be  necessary  slightly  to 
enlarge  the  opening.  The  blood  should  be  allowed  to  clot  completely, 
and  then,  by  means  of  a  sterile  platinum  needle,  the  clot  should  be  loosened 
from  the  sides  of  the  tube  in  order  that  it  may  freely  contract.  The  tube 
should  be  placed  in  the  ice-chest  till  the  following  morning,  when  the 
serum  can  be  pipetted  off  with  a  sterile,  nippled  pipette. 

Daily  samples  can  thus  be  obtained  from  an  animal.  If  care  be  taken 
not  to  make  ragged  openings  in  the  vein,  often  the  simple  removal 
of  the  previous  scab  will  be  followed  by  a  free  blood  flow. 

A  worker  associated  with  one  of  us  has  shown  that  this  method  can  be 
applied  in  guinea-pigs,  provided  these  be  of  fair  size.  Here  successive 
samples  of  2  c.c.  can  be  obtained  from  the  ear  veins. 

Fixation  of  Complement  or  Complement  Deviation. — From 
the  facts  given  above  it  follows  that  sensitised  corpuscles,  i.e. 
corpuscles  treated  with  immune-body,  may  be  made  to  serve  as  an 
indicator  for  the  presence  of  complement.  If  an  immune-body  is 
present  in  a  serum  heated  at  55°  C.,  the  serum  when  added  to  the 
corresponding  bacterium  leads  to  the  fixation  of  complement,  and 
thus  prevents  haemolysis  when  the  sensitised  corpuscles  are  added. 
If  we  represent  the  bacteria,  or  rather  the  receptors  in  the  bacteria, 
by  X,  the  immune-body  by  anti-X,  and  the  complement  by  C 


THE  SERUM  DIAGNOSIS  OF  SYPHILIS         131 

(normal  serum,  say  of  a  guinea-pig),  we  may  represent  the  method 
of  experiment  by  the  following  scheme  : — 


X  +  anti-X  +  C 


+  sensitised  corpuscles 


(The  vertical  dotted  line  represents  a  period  of  incubation  for 
one  and  a  half  hours  at  37°  C.) 

If  lysis  of  the  sensitised  corpuscles  does  not  occur  after  incuba- 
tion at  -37°  C.,  then  the  complement  has  been  fixed  and  an 
immune-body  has  been  shown  to  be  present,  provided  that  a 
suitable  control  shows  that  the  bacteria  alone,  without  immune- 
body,  do  not  fix  sufficient  complement  to  interfere  with  lysis. 

This  method  has  now  been  extensively  used  for  demonstrating 
the  presence  of  immune-bodies  in  the  blood  of  patients  suffering 
from  a  particular  bacterial  infection.  It  has  also  been  applied 
to  determine  whether  a  suspected  bacterium  is  really  the  cause 
of  a  disease,  for  if  the  bacterium  gives  with  the  serum  of  the 
patient  deviation  of  complement,  then  there  is  a  strong  pre- 
sumption that  it  is  the  infective  agent  (vide  Immunity). 

The  Serum  Diagnosis  of  Syphilis,  Wassermann  Reaction.— 
Wassernmim,  Neisser  and  Bruck,  proceeding  in  accordance  with 
the  facts  established  with  regard  to  the  deviation  of  complement, 
tested  whether  a  similar  phenomenon  might  not  be  obtained  in 
the  case  of  syphilis.  For  this  purpose  they  mixed  together  a 
watery  extract  of  syphilitic  liver,  rich  in  spirochaetes  (antigen), 
and  serum  from  a  syphilitic  case  (supposed  to  contain  anti-sub- 
stances), and  found  that  a  relatively  large  amount  of  complement 
was  fixed.  On  the  other  hand,  when  the  serum  from  a  non- 
syphilitic  case  was  substituted  for  the  syphilitic  serum,  little  or 
no  fixation  of  complement  occurred.  The  result  was  thus  in 
accordance  with  expectations  on  theoretical  grounds.  Marie 
and  Levaditi,  however,  found  that  an  extract  of  normal  guinea- 
pig's  liver  along  with  syphilitic  serum  fixed  complement,  and 
subsequent  observations  showed  that  extracts  of  other  tissues  are 
also  more  or  less  efficient,  as  are  also  certain  definite  substances, 
such  as  sodium  oleate,  sodium  glycocholate,  lecithin,  mixtures 
of  such  and  especially  mixtures  of  lecithin  and  cholesterin,  etc. 
Although  abundant  observations  have  established  the  validity 
of  the  test  as  a  means  of  diagnosis,  the  reaction  which  led  to  its 
discovery  is  no  longer  sufficient  to  explain  it,  and  the  nature  of 
the  reaction  is  not  yet  understood. 

In  order  to  carry  out  the  test,  we  require  (a)  serum  from  the 
suspected  case,  (6)  an  extract  of  liver  or  other  organ,  and 
(c)  the  fresh  serum  of  an  animal  to  act  as  complement.  The  fol- 
lowing are  the  details,  arranged  in  two  stages  : — 


132  METHODS  OF  EXAMINING  SERUM 


M 
(Jr 

.^ 

V^iJ  (t 
(y^        v 

w>*( 
fid***   act 


1.  We  add  to  a  small  test-tube  — 

(a)  '05  c.c.  of  serum  from  the  suspected  case,  heated  for  half 
an  hour  at  55°  C.  to  destroy  the  human  complement,  and  '5  c.c. 
of  "8  per  cent,  salt  solution  ; 

(b)  "I  c.c.  of  an  alcoholic  extract  of  guinea-pig's  or  ox's  liver 
(this  can  be  prepared  by  extracting  finely  minced  liver  with  four 
volumes  of  alcohol  for  3  to  4  days  and  then  filtering)  ; 

A  certain  amount  of  guinea-pig's  serum,  usually  '1  c.c.,  to 
act  as  complement. 

The  mixture  is  then  placed  in  the  incubator  for  one  and  a  half 
hours,  to  allow  fixation  of  complement  to  occur. 

2.  We  then  add  to  the  tube  1  c.c.  of  a  5  per  cent,  suspension 
of  sensitised  corpuscles  (usually  sheep's  or  ox's),  i.e.  corpuscles 
to  which  there  has  been  added  a  sufficient  quantity  of  immune 
serum  to  produce  lysis  on  the  addition  of  complement. 

The  mixture  is  then  placed  in  the  incubator  for  another  hour. 
If  lysis  of  the  corpuscles  does  not  occur,  the  complement  has 
been  fixed  in  the  first  stage  by  the  mixture  of  serum  and  liver 
extract.  This  is  a  positive  result,  and  indicates  the  presence  of 
syphilis.  If  the  corpuscles  undergo  lysis,  all  the  complement 
has  not  been  fixed  —  the  result  is  negative.  When  the  amount 
of  serum  to  be  tested  is  small,  the  amounts  given  may  all  be 
proportionately  reduced. 

Such  is  the  test  as  usually  performed,  and  in  this  form  it 
usually  gives  satisfactory  results.  It  is  to  be  noted,  however, 
on  the  one  hand,  that  the  liver  extract  alone  may  fix  a  certain 
amount  of  complement,  rarely  more  than  three  doses,  and,  on 
the  other  hand,  that  the  hsemolytic  value  of  fresh  serum  varies, 
i.e.  the  amount  of  complement  is  not  always  indicated  by  the 
volume  of  serum.  It  is  accordingly  better,  and  in  a  laboratory 
this  can  be  readily  done,  to  estimate  the  hamiolytic  dose  of  the 
guinea-pig's  serum,  and  to  prepare  a  series  of  tubes,  each  con- 
taining the  same  amounts  of  serum  and  of  liver  extract,  but 
with  a  different  number  of  doses  of  complement  in  each  tube.  In 
this  way  we  can  find  the  number  of  doses  of  complement  deviated 
in  each  case.  As  controls,  the  effect  of  the  extract  alone  and  of  the 
serum  alone  can  be  tested  at  the  same  time.  With  the  amounts 
of  extract  and  serum  mentioned,  a  positive  result  indicating  the 
presence  of  syphilis  may  be  accepted  when  five  or  more  doses  of 
complement  are  deviated  in  addition  to  the  amount  absorbed  in 
the  controls.  Some  observers  use  the  same  amount  of  comple- 
ment in  each  tube,  but  vary  the  amounts  of  suspected  serum,  and 
in  this  way  some  idea  of  the  deviating  power  of  the  serum  is 
obtained,  but  we  consider  that  the  method  given  is  to  be  preferred. 


WRIGHT'S  METHOD  OF  COUNTING  BACTERIA     133 

THE  PREPARATION  OF  VACCINES. 

During  recent  years,  in  consequence  of  the  work  of  Sir 
Almroth  Wright,  the  principle  of  treating  bacterial  disease  by 
vaccines  has  been  very  much  developed.  The  general  principle 
is  to  inject  into  the  infected  individual  an  emulsion  of  dead 
bacteria.  In  certain  cases  the  bacteria  are  subjected  to  dis- 
integrating processes  before  being  used,  but  most  frequently  the 
vaccines  simply  contain  killed  bacterial  cells,  and  the  preparation 
is  comparatively  simple. 

In  the  case  of  pyoyenic  cocci,  either  bouillon  cultures  or  a 
growth  off  sloped  agar  emulsified  in  normal  saline  is  taken  and 
killed  by  heat.  The  temperature  employed  should  be  the 
minimum  at  which  death  occurs,  say  65°  C.,  applied  for  half  an 
hour.  In  the  case  of  certain  staphylococci,  we  have  found, 
however,  that  a  higher  temperature  is  necessary.  After  any 
sterilisation  procedure,  tubes  of  agar  must  be  inoculated  from 
the  presumably  dead  vaccine,  and  incubated  for  twenty-four 
hours  in  order  to  ascertain  if  the  sterilisation  has  been  effective. 
As  the  dosage  of  a  vaccine  is  of  great  importance,  it  is  necessary 
to  count  the  bacteria  present.  This  is  done  by  one  of  the 
methods  given  below.  Appropriate  doses  (see  Chapter  VII.)  are 
then  with  all  aseptic  precautions  measured  by  means  of  a  sterile 
graduated  pipette,  and  placed,  along  with  an  equal  volume  of 
•5  per  cent,  lysol,  in  little  glass  bulbs  drawn  out  to  a  capillary 
tube  at  one  end.  These  when  charged  are  sealed  off,  and  for 
use  the  sealed  end  is  broken  off,  the  contents  are  sucked  up  into 
a  sterile  hypodermic  needle,  and  injected  fairly  deeply  into  the 
-kin,  usually  in  the  region  of  the  flank. 

In  the  case  of  the  typhoid  bacillut,  organisms  are  used  of  such 
virulence  that  a  quarter  of  a  twenty-four  hours'  old  sloped  agar 
culture,  when  administered  hypodermically,  will  kill  a  guinea- 
pig  of  from  350  to  400  grams.  Flasks  of  bouillon  are  inoculated 
with  such  a  culture  for  forty-two  hours  at  37°  C.  The  bacteria  are 
then  killed  by  the  flask  being  put  into  a  water  bath  at  62°  C. 
for  fifteen  minutes  ;  '5  per  cent,  lysol  is  added,  and  the  bacteria 
in  the  vaccine  are  counted.  By  such  methods,  vaccines  against 
any  of  the  pyogenic  cocci  and  against  any  members  of  the  coli- 
typhoid  group  can  be  made. 

The  vaccines  used  in  tuberculosis,  cholera,  and  plague  will  be 
described  in  the  chapters  on  these  diseases. 

Wright's  Method  of  counting  the  Bacteria  in  Dead 
Cultures. — In  the  making  of  vaccines  it  is,  as  indicated  above, 
necessary  to  know  the  total  number  of  bacterial  cells,  whether 


134  THE  PREPARATION  OF  VACCINES 

dead  or  living,  present  in  a  culture,  for  the  dead  as  well  as  the 
living  contain  the  toxins  which  may  stimulate  the  therapeutic 
capacities  of  the  body.  The  method  consists  in  making  a 
mixture  of  blood  (whose  content  in  red  blood  corpuscles  is 
known)  with  the  bacterial  culture,  and  comparing  the  number  of 
bacteria  with  the  number  of  corpuscles.  The  observer  first 
estimates  the  red  cells  in  his  blood  ;  a  capillary  pipette  with  a 
rubber  nipple  and  with  a  mark  near  its  capillary  extremity  is 
then  taken,  blood  is  sucked  up  to  the  mark,  then  an  air-bubble, 
and  then  an  equal  volume  of  the  bacterial  emulsion  diluted 
according  to  the  empirical  estimate  the  observer  forms  of  its 
strength.  The  blood  and  bacterial  emulsion  are  then  thoroughly 
mixed  by  being  drawn  backwards  and  forwards  in  the  wide 
part  of  the  pipette,  a  drop  is  blown  out  on  to  a  slide,  and  a 
blood  film  is  spread  which  may  be  stained  by  Leishman's 
method.  The  bacteria  and  blood  corpuscles  are  now  separately 
enumerated  in  a  series  of  fields  in  different  parts  of  the 
preparation.  If  a  dilution  has  been  taken  in  which  a  large 
number  of  bacteria  are  present,  an  artificial  field  may  be  used, 
made  by  drawing  with  the  oil  pencil  a  small  square  on  a  circular 
cover-glass,  and  dropping  the  latter  on  to  the  diaphragm  of  the 
microscope  eye-piece.  Suppose,  now,  that  the  observer's  blood 
contained  5,000,000  red  cells  per  c.mm.,  that  to  the  bacterial 
emulsion  three  volumes  of  diluent  had  been  added,  and  that  in 
the  fields  examined  there  were  500  red  cells  and  600  bacteria. 
It  is  evident  that  in  the  undiluted  culture  for  500  red  cells  there 
would  have  been  2400  bacteria.  Now  500  :  2400  : ;  5,000,000  : 
24,000,000,  which  last  figure  is  the  number  of  bacteria  per 
c.mm.  of  the  emulsion. 

It  has  been  found  in  the  case  of  certain  bacteria,  e.g.  the 
members  of  the  coli-typhoid  and  cholera  groups,  that  when  an 
emulsion  of  these  is  mixed  with  whole  blood,  the  serum  of  the 
latter  may  have  a  bacteriolytic  or  an  agglutinating  action  on  the 
organisms,  which  interferes  with  the  counting.  To  obviate  the 
inaccuracies  or  difficulties  thus  introduced,  Harrison  has  modified 
Wright's  method  by  substituting,  in  a  given  quantity  of  blood, 
normal  saline  for  the  serum.  The  method  is  as  follows  : — A 
capillary  pipette  has  a  mark  made  upon  it,  to  which  blood  is 
sucked  up  and  quickly  expelled  into  a  small  tube  containing  a 
little  "75  per  cent,  sodium  citrate  solution  ;  any  remaining  blood  is 
washed  out  of  the  pipette  with  the  same  fluid.  The  tube  is  then 
centrifuged  to  deposit  the  corpuscles,  the  supernatant  fluid 
carefully  removed,  and  the  corpuscles  are  washed  by  centrifuging 
twice  or  thrice  with  normal  saline,  care  being  always  taken  not 


WRIGHT'S  METHOD  OF  COUNTING  BACTERIA     135 

to  lose  any  of  the  corpuscles  in  the  successive  washings.  After 
the  last  washing  the  corpuscles  are  sucked  up  into  the  pipette, 
and  the n  saline  up  to  the  mark  which  indicated  the  volume  of 
the  original  blood.  Such  a  mixture  is  taken,  and,  to  prevent 
loss  of  corpuscles,  the  pipette  and  tubes  are  washed  with  a 
definite  number  of  equal  volumes  of  broth  or  saline.  Thus 
there  can  be  obtained  in  a  watch-glass  a  mixture  of,  say,  one 
volume  of  corpuscles  and  saline,  and  two  volumes  of  the  diluting 
fluid.  To  this  mixture  is  now  added  an  appropriate  number  of 
volumes,  again  measured  in  the  same  pipette,  of  the  bacterial 
emulsion  to  be  counted,  the  amount,  of  course,  depending  upon 
a  rough  judgment  which  with  experience  can  be  made  of  the 
probable  numbers  present.  A  drop  of  the  mixture  is  put  under 
a  cover-glass,  and  the  numbers  of  corpuscles  on  the  one  hand  and 
of  bacteria  on  the  other  present  in  a  number  of  fields  are 
counted.  It  is  not  necessary  to  stain  the  bacteria,  but  in  the 
case  of  motile  organisms  it  is  recommended  that  they  be 
rendered  motionless  by  using  as  a  diluent  saline  to  which  formol 
has  been  added  in  the  proportion  of  two  or  three  drops  to  10  c.c. 
If  the  number  of  red  blood  corpuscles  in  the  observer's  blood  be 
known,  it  is  evident  that  the  amount  of  blood  corresponding  to 
a  certain  number  of  blood  corpuscles  in  a  microscopic  field  can 
be  calculated,  and  the  number  of  bacteria  present  in  the  same 
amount  of  the  mixture  will  be  the  number  corresponding  to  the 
number  of  corpuscles.  Thus  it  is  now  only  necessary  to  allow 
for  the  dilution  to  obtain  the  number  of  bacteria  in  the  original 
emulsion. 

GENERAL  BACTERIOLOGICAL  DIAGNOSIS. 

Under  this  heading  we  have  to  consider  the  general  routine 
which  is  to  be  observed  by  the  bacteriologist  when  any  material 
is  submitted  to  him  for  examination.  The  object  of  such 
examination  may  be  to  determine  whether  any  organisms  are 
present,  and  if  so,  what  organisms ;  or  the  bacteriologist  may 
simply  be  asked  whether  a  particular  organism  is  or  is  not 
present.  In  any  case,  his  inquiry  must  consist  (1)  of  a  micro- 
scopic examination  of  the  material  submitted ;  (2)  of  an  attempt 
to  isolate  the  organisms  present ;  and  (3)  of  the  identification  of 
the  organisms  isolated.  We  must,  however,  before  considering 
these  points,  look  at  a  matter  often  neglected  by  those  who  seek 
a  bacteriological  opinion,  namely,  the  proper  methods  of  ob- 
toiniii'i  and  transferring  to  t/ie  bacteriologist  the  material  u'hich 
I i'  /.s  to  be  asked  to  examine.  The  general  principles  here  are 


136     GENERAL  BACTERIOLOGICAL  DIAGNOSIS 


(1)  that   every   precaution   must   be   adopted   to    prevent   the 
material  from  being  contaminated  with  extraneous  organisms; 

(2)  that  nothing  be  done  which  may  kill  any  organisms  which 
may  be  proper  to  the  inquiry  ;  and  (3)  that  the  bacteriologist 
obtain  the  material  as  soon  as  possible  after  it  has  been  removed 
from  its  natural  surroundings. 

The  sources  of  materials  to  be  examined,  even  in  patho- 
logical bacteriology  alone,  are,  of  course,  so  varied  that  we  can 
but  mention  a  few  examples.  It  is,  for  instance,  often  necessary 
to  examine  the  contents  of  an  abscess.  Here  the  skin  must  be 
carefully  purified  by  the  usual  surgical  methods  •  the  knife  used 
for  the  incision  is  preferably  to  be  sterilised  by  boiling ;  the  first 
part  of  the  pus  which  escapes  is  allowed  to  flow  away  (as  it  might 
be  spoilt  by  containing  some  of  the  antiseptics  used  in  the 
purification),  and  a  little  of  what  subsequently  escapes  allowed 
to  flow  into  a  sterile  test-tube.  If  test-tubes  sterilised  in  a 
laboratory  are  not  at  hand,  an  ordinary  test- 
tube  may  be  quarter-filled  with  water  and 
vigorously  boiled  over  a  spirit-lamp.  The  tube 
is  then  emptied  and  plugged  with  a  plug  of 
cotton  wool,  the  outside  of  which  has  been 
singed  in  a  flame.  Small  stoppered  bottles 
may  be  sterilised  and  used  in  the  same  way. 
A  discharge  to  be  examined  may  be  so  small 
in  quantity  as  to  make  the  procedure  described 
impracticable.  It  may  be  caught  on  a  piece 
of  sterile  plain  gauze,  or  of  plain  absorbent 
wool,  which  is  then  placed  in  a  sterile  vessel. 
Wool  or  gauze  used  for  this  purpose,  or  for 
swabbing  out,  say  the  throat,  to  obtain  shreds 
of  suspicious  matter,  must  have  no  antiseptic 
impregnated  in  it,  as  the  latter  may  kill  the 
bacteria  present  and  make  the  obtaining  of 
cultures  impossible. 

Fluids   from  the   body  cavities,  urine,  etc., 

FIG.  46.— Test-tube   mav  j^  secured  with  sterile  pipettes.     To  make 

and    pipette     ar-          J     £     ,  .       _    .      .          rf         ,.  .,, 

ranged  for  obtain-    one  o*  these,  take  9  inches  of  ordinary  quill 

ing  fluids  contain-   glass-tubing,  draw  out  one  end  to  a  capillary 

diameter,  and  place  a  little  plug  of  cotton  wool 

in    the   other  end.      Insert   this    tube  through 

the  cotton  plug  of  an  ordinary  test-tube,  and  sterilise  by  heat. 

To  use  it,  remove  test-tube  plug  with  the  quill  tube  in  its  centre, 

suck  up  some  of  the    fluid   into  the  latter,  and  replace  in  its 

former  position    in  the  test-tube   (Fig.   46).      Another  method 


KOUTINE  EXAMINATION  OF  MATERIAL       137 

very  convenient  for  transport  is  to  make  two  constrictions  on 
the  glass  tube  at  suitable  distances,  according  to  the  amount  of 
fluid  to  be  taken.  The  fluid  is  drawn  up  into  the  part  between 
the  constrictions,  but  so  as  not  to  fill  it  completely.  The  tube 
is  then  broken  through  at  both  constrictions,  and  the  thin  ends 
an-  sealed  by  heating  in  a  flame. 

Solid  organs  to  be  examined  should,  if  possible,  be  obtained 
whole.  They  may  be  treated  in  one  of  two  ways.  (1)  The 
surface  over  one  part  about  an  inch  broad  is  seared  with  a 
cautery  heated  to  dull  red  heat.  All  superficial  organisms  are 
thus  killed.  An  incision  is  made  in  this  seared  zone  with  a 
sterile  scalpel,  and  small  quantities  of  the  juice  are  removed  by 
a  platinum  spud  to  make  cover-glass  preparations  and  plate 
or  smear  cultures.  (2)  An  alternative  method  is  as  follows  : — 
The  surface  is  sterilised  by  soaking  it  well  with  1  to  1000 
corrosive  sublimate  for  half  an  hour.  It  is  then  dried,  and  the 
capsule  of  the  organ  is  cut  through  with  a  sterile  knife,  the 
incision  being  further  deepened  by  tearing.  In  this  way  a 
perfectly  uncontaminated  surface  is  obtained.  Hints  are  often 
obtained  from  the  clinical  history  of  the  case  as  to  what  the 
procedure  ought  to  be  in  examination.  Thus,  as  a  matter  of 
practice,  cultures  of  tubercle  and  often  of  glanders  bacilli  can 
be  easily  obtained  only  by  inoculation  experiments.  Typhoid 
bacilli  need  hardly  be  looked  for  in  the  faeces  after  the  first  ten 
days  of  the  disease,  and  so  on. 

Eoutine  Procedure  in  Bacteriological  Examination  of 
Material. — In  the  case  of  a  discharge  regarding  which  nothing 
is  known,  the  following  procedure  should  be  adopted : — 
(1)  Several  cover-glass  preparations  should  be  made.  One  ought 
to  be  stained  with  saturated  watery  methylene-blue,  one  with 
a  stain  containing  a  mordant  such  as  Ziehl-Neelsen  carbol- 
fuchsin,  one  by  Gram's  method.  (2)  a.  Gelatin  plates  should 
be  made  and  kept  at  room  temperature ;  b.  a  series  of  agar 
plates  or  successive  strokes  on  agar  tubes  (p.  60)  should  be  made 
and  incubated  at  37°  C.  Method  b  of  course  gives  results 
more  quickly.  In  every  case  when  an  unknown  disease  is 
being  investigated,  some  of  the  material  should  be  subjected  to 
methods  suitable  to  the  growth  of  anaerobic  bacteria.  If  micro- 
scopic investigation  reveals  the  presence  of  bacteria,  it  is  well 
to  keep  the  material  in  a  cool  place  till  next  day,  when,  if  no 
growth  has  appeared  in  the  incubated  agar,  some  other  culture 
medium  (e.y.  blood  serum  or  agar  smeared  with  blood)  may  be 
employed.  If  growth  has  taken  place,  say  in  the  agar  plates, 
one  with  about  two  hundred  or  fewer  colonies  should  be  made 


138     GENERAL  BACTERIOLOGICAL  DIAGNOSIS 

the  chief  basis  for  research.  In  such  a  plate  the  first  question 
to  be  cleared  up  is  :  Do  all  the  colonies  present  consist  of  the 
same  bacterium  1  The  shape  of  the  colony,  its  size,  the  appear- 
ance of  the  margin,  the  graining  of  the  substance,  its  colour, 
etc.,  are  all  to  be  noted.  One  precaution  is  necessary,  namely, 
it  must  be  noted  whether  the  colony  is  on  the  surface  of  the 
medium  or  in  its  substance,  as  colonies  of  the  same  bacterium 
may  exhibit  differences  according  to  their  position.  The 
arrangement  of  the  bacteria  in  a  surface  colony  may  be  still 
more  minutely  studied  by  means  of  impression  preparations. 
A  cover-glass  is  carefully  cleaned  and  sterilised  by  passing 
quickly  several  times  through  a  Bunsen  flame.  It  is  then  placed 
on  the  surface  of  the  medium,  and  gently  pressed  down  on  the 
colony.  The  edge  is  then  raised  by  a  sterile  needle,  it  is  seized 
with  forceps,  dried  high  over  the  flame,  and  treated  as  an 
ordinary  cover-glass  preparation.  In  this  way  very  characteristic 
appearances  may  sometimes  be  noted  and  preserved,  as  in  the 
case  of  the  anthrax  bacillus.  The  colonies  on  a  plate  having 
been  classified,  a  microscopic  examination  of  each  group  may 
be  made  by  means  of  cover-glass  preparations,  and  tubes  of 
gelatin  and  agar  are  inoculated  from  each  representative  colony. 
Each  of  the  colonies  used  must  be  marked  for  future  reference, 
preferably  by  drawing  a  circle  round  it  on  the  under  surface  of 
the  plate  or  capsule  with  one  of  Faber's  pencils  for  marking  on 
glass,  a  number  or  letter  being  added  for  easy  reference. 

The  general  lines  along  which  observation  is  to  be  made 
in  the  case  of  a  particular  bacterium  may  be  indicated  as 
follows  : — 

1.  Microscopic  Appearances. — For  ordinary  descriptive  pur- 
poses, young  cultures,  say  of  twenty-four  hours'  growth,  on  agar 
should  be  used,  though  appearances  in  older  cultures,  such  as 
involution  forms,  etc.,  may  also  require  attention.  Note,— (1) 
the  form  ;  (2)  the  size  ;  (3)  the  appearance  of  the  protoplasmic 
contents,  especially  as  regards  uniformity  or  irregularity  of 
staining ;  (4)  the  method  of  grouping ;  (5)  the  staining  reactions. 
Has  it  a  capsule  1  Does  the  bacterium  stain  with  simple  watery 
solutions'?  Does  it  require  the  use  of  stains  containing 
mordants  ?  How  does  it  behave  towards  Gram's  method  ?  It 
is  important  to  investigate  the  first  four  points,  both  wrhen  the 
organism  is  in  the  fluids  or  tissues  of  the  body  and  when  growing 
in  artificial  media,  as  slight  variations  occur.  It  must  also  be 
borne  in  mind  that  slight  variations  are  observed  according  to 
the  kind  and  consistence  of  the  medium  in  which  the  organism 
is  growing.  (6)  Is  it  motile,  and  has  it  fiagella?  If  so,  how 


GROWTH  CHARACTERISTICS  139 

are  they  arranged  1  (7)  Does  it  form  spores,  and  if  so,  under 
what  conditions  as  to  temperature,  etc.1? 

'2.  fi'roii'tk  Characteristics. — Here  the  most  important  points 
on  which  information  is  to  be  asked  are,  What  are  the  characters 
of  UTO  \\th  and  what  are  the  relations  of  growth  (l)to  tempera- 
ture ;  (2)  to  oxygen  ?  These  can  be  answered  from  some  of  the 
folloNsin-  experiments  : — 

A.  (Jrowth  on  gelatin.  (1)  Stab  culture.  Note, — (a)  rate  of 
growth  ;  (/>)  form  of  growth,  (a)  on  surface,  (/?)  in  substance;  (c) 
presence  or  absence  of  liquefaction  ;  (d)  colour;  (e)  presence  or 
absence  of  gas  formation  and  of  characteristic  smell ;  (/)  relation 
to  reaction  of  medium.  (2)  Streak  culture.  (3)  Shake  culture. 
(  \ )  Plate  cultures.  Note  ap}>earances  of  colonies,  (a)  superficial, 
(b)  deep.  (5)  Growth  in  fluid  gelatin  at  37°  C. 

U.  (Jrowth  on  agar  at  37°  C.  (1)  Stab.  (2)  Streak.  Also 
on  glycerin-agar,  blood-agar,  etc.  Appearances  of  colonies  in 
airar  plates. 

C.  Growth   in  bouillon,   (a)    character   of   growth,   (£)  smell, 
('•)  reaction. 

D.  Growth  on    social   media.     (1)    Solidified   blood  serum. 
(2)     Potatoes.     (3)    Lactose   and    other    sugar    media.     Does 
fermentation  occur,  and  is  gas  formed  ?     (4)  Milk.     Is  it  curdled 
or  turned  sour  ?     (5)  Litmus  media.     Note  changes  in  colour. 
(6)  Peptone  solution.     Is  indol  formed  1 

K.  What  is  the  viability  of  organism  on  artificial  medial 
3.  Result*  <>>'  inoculation  experiment*  on  animals. 
By  attention  to  such  points  as  these  a  considerable  knowledge 
is  attained  regarding  the  bacterium,  which  will  lead  to  its 
identification.  In  the  case  of  many  well-known  organisms, 
however,  a  few  of  the  above  points  taken  together  will  often 
be  sufficient  for  the  recognition  of  the  species,  and  experience 
teaches  what  are  the  essential  points  as  regards  any  individual 
organism.  In  the  course  of  the  systematic  description  of  the 
pathogenic  organisms,  it  will  be  found  that  all  the  above  points 
will  be  referred  to,  though  not  in  every  case. 

The  methods  hy  wliicli  the  morphological  and  biological  characteristics 
of  any  growth  may  be  observed  have  already  been  fully  described.  It 
need  cnly  l.r  pointed  out  here  that  in  giving  descriptions  of  bacteria  the 
greatest  care  must  be  taken  to  state  every  detail  of  investigation.  Thus 
in  any  description  of  microscopic  appearances  the  age  of  the  growth  from 
which  tin-  preparation  was  made,  the  medium  employed,  the  temperature 
at  \\liidi  development  took  place,  must  be  noted,  along  with  the  stain 
which  was  used  ;  and  with  regard  to  the  latter  it  is  always  preferable  to 
employ  one  of  the  well-known  staining  combinations,  such  as  Loffler's 
methylene-blue.  Especial  care  is  necessary  in  stating  the  size  of  a 


140     GENERAL  BACTERIOLOGICAL  DIAGNOSIS 

bacterium.  The  apparent  size  often  shows  slight  variations  dependent 
on  the  stain  used  and  the  growth  conditions  of  the  culture.  Accurate 
measurements  of  bacteria  can  only  be  made  by  preparing  microphoto- 
graphs  of  a  definite  magnification,  and  measuring  the  sixes  on  the 
negatives.  From  these  the  actual  sizes  can  easily  be  calculated.  A 
rough  method  of  estimating  the  size  of  an  organism  is  to  mix  a  little 
with  a  drop  of  the  observer's  blood  and  make  a  blood  film.  As  the  size 
of  a  normal  red  blood  corpuscle  is  about  7 '5  IJL,  an  idea  of  the  size  of  a 
bacterium  can  be  obtained  by  comparing  it  with  this  as  a  standard.  In 
describing  bacterial  cultures  it  must  be  borne  in  mind  that  the  appearances 
often  vary  with  the  age.  It  is  suggested  that  in  the  case  of  cultures 
grown  at  from  36°  to  37°  C.  the  appearances  between  twenty-four  and 
forty-eight  hours  should  be  made  the  basis  of  description,  and  in  the 
case  of  cultures  grown  between  18°  and  22°  C.  the  appearances  between 
forty-eight  and  seventy-two  hours  should  be  employed.  The  culture 
fluids  used  must  be  made  up  and  neutralised  by  the  precise  methods 
already  described.  The  investigator  must  give  every  detail  of  the 
methods  he  has  employed,  in  order  that  his  observations  may  be  capable 
of  repetition. 

In  the  case  of  a  number  of  pathogenic  organisms,  identification 
is  a  comparatively  easy  matter.  In  some  cases,  however,  great 
difficulties  arise  in  consequence  of  the  existence  of  groups  of 
organisms  presenting  closely  allied  characters,  and  the  difficulty 
and  importance  of  identification  is  enhanced  by  the  fact  that 
the  same  group  may  include  both  harmful  and  innocent  members. 
Examples  of  this  occurrence  are  found  in  the  pyogenic  cocci  and 
their  allies,  in  the  coli-typhoid  group  of  bacilli,  and  in  the  group 
of  cholera  vibrios.  In  such  cases  it  is  usually  necessary  to  take 
into  account  all  the  morphological  and  cultural  reactions  of  an 
organism  before  it  can  be  adequately  classified.  Within  recent 
years  attempts  have  been  made  to  apply  the  statistical  method 
to  the  solution  of  the  difficulties  of  the  situation,  and  here  the 
results  appear  to  be  promising.  The  method  has  been  applied 
to  the  coccaceae  by  Winslow  and  Rogers,  who  have  investigated 
500  strains  of  cocci  isolated  from  the  tissues  in  disease,  from 
the  outer  surfaces  of  the  normal  human  body,  from  water,  earth, 
and  air.  A  great  variety  of  properties  was  studied,  and  while 
in  each  test  applied  wide  variation  was  exhibited  in  such  bacteria, 
there  usually  emerged  a  type  property  to  which  individual 
strains  tended  to  approach.  Thus,  while  the  size  varies  from 
"1  to  2'0  fji,  out  of  about  350  strains  examined  about  115 
measured  '3  ^  and  the  remaining  strains  tended  to  be  a  little 
below  or  a  little  above  this  figure.  When  similar  lines  of 
inquiry  were  pursued  with  regard  to  other  characteristics  of 
the  organisms,  it  was  found  that  important  correlations  could 
be  noted.  Thus  capacity  for  staining  by  Gram's  method  was 
found  especially  amongst  the  staphylococci  and  streptococci  as 


INOCULATION  OF  ANIMALS  HI 

contrasted  with  forms  tending  to  grow  in  sarcinal  packets,  and 
the  Gram-staining  forms  were  chiefly  parasitic  in  habitat. 
Looking  at  their  results  as  a  whole,  Winslow  and  Rogers  divide 
the  cocci  into  two  great  groups,  the  Paracoccaceae  and  the  Meta- 
coccaceae.  The  former  comprise  most  of  the  forms  derived  from 
the  body,  show  a  staphylococcal  or  streptococcal  tendency,  stain 
by  Gram,  yield  only  moderate  surface  growths,  form  acid  in 
carbohydrates,  and  produce  no  pigment  or  a  white  or  orange 
colour.  The  latter  come  chiefly  from  air  and  water,  often  are 
suviniform,  decolorise  by  Gram,  grow  well  on  the  surface  of 
media,  do  not  ferment  carbohydrates,  and  produce  red  or  yellow 
pigment.  On  similar  lines,  further  subdivision  of  the  groups 
could  be  effected.  It  is  manifest  that  important  means  of 
differentiating  allied  bacteria  may  be  available  by  the  extended 
application  of  this  method. 

INOCULATION  OF  ANIMALS. l 

The  animals  generally  chosen  for  inoculation  are  the  mouse, 
the  rat,  the  guinea-pig,  the  rabbit,  and  the  pigeon.  Great  caution 
must  be  shown  in  drawing  conclusions  from  isolated  experiments 
on  rabbits,  as  these  animals  often  manifest  exceptional  symptoms, 
and  are  very  easily  killed.  Dogs  are,  as  a  rule,  rather  insusceptible 
to  microbic  disease,  and  the  larger  animals  are  too  expensive  for 
ordinary  laboratory  purposes.  In  the  case  of  the  mouse  and  rat 
the  variety  must  be  carefully  noted,  as  there  are  differences  in 
>iisreptibility  between  the  wild  and  tame  varieties,  and  between 
the  white  and  In-own  varieties  of  the  latter.  In  the  case  of  the 
wild  varieties,  these  must  be  kept  in  the  laboratory  for  a  week  or 
two  before  use,  as  in  captivity  they  are  apt  to  die  from  very  slight 
causes  ;  and,  further,  each  individual  should  be  kept  in  a  separate 
cage,  as  they  show  great  tendencies  to  cannibalism.  Of  all  the 
ordinary  animals  the  most  susceptible  to  microbic  disease  is  the 
guinea-pig.  Practically  all  inoculations  are  performed  by  means 
of  the  hypodermic  syringe.  The  best  variety  is  made  on  the 
ordinary  model  with  metal  mountings,  asbestos  washers,  and 
preferably  furnished  with  platinum-indium  needles.  Before  use, 
the  syringe  and  the  needle  are  sterilised  by  boiling  for  five 
minutes.  The  materials  used  for  inoculation  are  cultures,  animal 
•  •\ IK lat  ions,  or  the  juice  of  organs.  If  the  bacteria  already  exist 
in  a  fluid  there  is  no  difficulty.  The  syringe  is  most  conveniently 
filled  out  of  a  shallow  conical  test-glass,  which  ought  previously 

1  Experiments  on  animals,  of  course,  cannot,  in  Britain,  be  performed  with- 
out a  licence  granted  by  the  Home  Secretary. 


142  INOCULATION  OF  ANIMALS 

to  have  been  covered  with  a  cover  of  filter  paper  and  sterilised. 
If  an  inoculation  is  to  be  made  from  organisms  growing  on  the 
surface  of  a  solid  medium,  either  a  little  ought  to  be  scraped 
off  and  shaken  up  in  sterile  bouillon  or  '85 ,  per  cent,  salt 
solution  to  make  an  emulsion,  or  a  little-  sterile  fluid  is  poured 
on  the  growth,  and  the  latter  scraped  off  into  it.  This  fluid  is 
then  filtered  into  the  test-glass  through  a  plug  of  sterile  glass 
wool.  This  is  easily  effected  by  taking  a  piece  of  f-inch  glass- 
tubing  3  inches  long,  drawing  one  end  out  to  a  fairly  narrow  point, 
plugging  the  tube  with  glass  wool  above  the  point  where  the 
narrowing  commences,  and  sterilising  by  heat.  By  filtering  an 
emulsion  through  such  a  pipette,  flocculi  which  might  block  the 
needle  are  removed.  If  a  solid  organ  or  an  old  culture  is  used 
for  inoculation,  it  ought  to  be  rubbed  up  in  a  sterile  porcelain  or 
metal  crucible  with  a  little  sterile  distilled  water,  by  means  of  a 
sterile  glass  rod,  and  the  emulsion  filtered  as  in  the  last  case. 

The  methods  of  inoculation  generally  used  are:  (1)  by  scari- 
fication of  the  skin ;  (2)  by  subcutaneous  injection ;  (3)  by 
intraperitoneal  injection ;  (4)  by  intravenous  injection ;  (5)  by 
injections  into  special  regions,  such  as  the  anterior  chamber  of 
the  eye,  the  substance  of  the  lung,  etc.  Of  these  (2)  and  (3) 
are  most  frequently  used.  When  an  anaesthetic  is  to  be  ad- 
ministered, this  is  conveniently  done  by  placing  the  animal, 
along  with  a  piece  of  cotton  wool  or  sponge  soaked  in  chloroform, 
under  a  bell-jar  or  inverted  glass  beaker  of  suitable  size. 

1.  Scarification. — A  few  parallel  scratches  are  made  in  the 
skin  of  the  abdomen  previously  cleansed,  just  sufficiently  deep 
to  draw  blood,  and  the  infective  material  is  rubbed  in  with  a 
platinum  eyelet.     The  disadvantage  of  this  method  is  that  the 
inoculation  is  easily  contaminated.     The  method  is  only  occasion- 
ally used. 

2.  Subcutaneous  Injection. — A  hypodermic  syringe  is  charged 
with  the  fluid  to  be  inoculated.     The  hair  is  cut  off  the  part  to 
be  inoculated,  and  the  skin  purified  with  1   to  1000  corrosive 
sublimate,  or  by  dropping  upon  it  some  strong  solution  of  iodine. 
The  skin  is  then  pinched  up,  and,  the  needle  being  inserted,  the 
requisite  dose  is  administered.     The  wound  is  then  sealed  with  a 
little  collodion. 

3.  Intraperitoneal    Injection.  —  This    may  be    performed  by 
means  of  a  special  form  of  needle.     The  needle  is  curved,  and 
has  its  opening  not  at  the  point,  but  in  the  side  in  the  middle 
of  the  arch  (Fig.   47).      The  hair  over  the  lower  part  of  the 
abdomen  is  cut,  and  the  skin  purified  with  an  antiseptic.     The 
whole  thickness  of   the    abdominal  walls    is    then   pinched  up 


METHODS  OF  INOCULATION 


143 


FIG.  47.  — Hollow 
needle  with 
lateral  aperture 
(at  a)  for  intra- 
jifi'iloneal  in- 
oculatious. 


between  the  forefingers   and   thumbs  of    the  two  hands,   and 
the    needle   is   plunged    through   the  fold  thus   formed.      The 
result  is  that  the  hole  in  the  side  of  the  needle  is  within  the 
abdominal  cavity,  and  the  inoculation  can  thus 
In-  made.     Intraperitoneal  inoculation  can  also 
be    practised    with    an    ordinary    needle.     The 
mode   of   procedure    is   similar,    but,   after   the 
needle  is  plunged  through  the  abdominal  fold, 
it  is  partially  withdrawn  till  the   point  is  felt 
to  be  free  in  the  peritoneal  cavity,  when  the 
injection  is  made.     There  is  little  risk  of  injur- 
ing the  intestines  by  either  method. 

4.  Intravenous    Injection. — The    vein    most 
usually  chosen  is  one   of   the   auricular  veins. 
Tli<-    part   has  the   hair   removed,  the   skin    is 
purified,    and    the    vein    made    prominent    by 
pressing  on  it  between  the  point  of  inoculation 
and  the  heart.     The  needle  is  then  plunged  into 
the  vein,  and  the  fluid  injected.     That  it  has 
perforated   the    vessel    will    be    shown    by    the 
escape  of  a  little  blood ;  and  that  the  injection 
has  taken  place  into  the  lumen  of  the  vessel  will  be  known  by 
the  absence  of  the  small  swelling  which  occurs  in  subcutaneous 
injections.     If  preferred,   the   vein  may  be    first   laid  bare   by 
snipping  the  skin  over  it.     The  needle  is  then  introduced. 

"».  inoculation  into  the  Anterior  Gliamber  of  the  Eye. — Local 
anaesthesia  is  established  by  applying  a  few  drops  of  2  per  cent, 
solution  of  hydrochlorate  of  cocaine.  The  eye  is  fixed  by  pinch- 
ing up  the  orbital  conjunctiva  with  a  pair  of  fine  forceps,  and, 
the  edge  of  the  cornea  being  perforated  by  the  hypodermic  needle, 
the  injection  is  easily  accomplished. 

Sometimes  inoculations  are  made  by  planting  small  pieces  of 
pathological  tissues  in  the  subcutaneous  tissue.  This  is  especially 
dune  in  the  case  of  glanders  and  tubercle.  The  skin  over  the 
back  is  purified,  and  the  hair  cut.  A  small  incision  is  made  with 
a  sterile  knife,  and  the  skin  being  separated  from  the  subjacent 
tissues  by  means  of  the  ends  of  a  blunt  pair  of  forceps,  a  little 
pocket  is  formed  into  which  a  piece  of  the  suspected  tissue  is 
inserted.  The  wound  is  then  closed  with  a  suture,  and  collodion 
is  applied.  In  the  case  of  guinea-pigs,  the  abdominal  wall  is  to 
br  preferred  as  the  site  of  inoculation,  as  the  skin  over  the  back 
is  extremely  thick. 

Injections  are  sometimes  made  into  other  parts  of  the  body, 
'.'/.  the  pleurae,  the  cranium,  the  spinal  canal.  With  regard  to 


144  INOCULATION  OF  ANIMALS 

the  last,  Ford-Robertson  has  pointed  out  that  in  the  rabbit  it 
can  be  easily  practised  through  the  space  between  the  seventh 
lumbar  and  first  sacral  vertebrae.  The  spine  of  the  former 
lies  in  a  line  with  the  iliac  crests.  With  regard  to  operative 
procedures  in  special  regions  of  the  body,  it  is  unnecessary  to 
describe  these,  as  the  application  of  the  general  principles 
employed  above,  together  with  those  of  modern  aseptic  surgery, 
will  sufficiently  guide  the  investigator  as  to  the  technique  which 
is  requisite. 

After  inoculation,  the  animals  ought  to  be  kept  in  comfortable 
cages,  which  must  be  capable  of  easy  and  thorough  disinfection 
subsequently.  For  this  purpose  galvanised  iron  wire  cages  are 
the  best.  They  can  easily  be  sterilised  by  boiling  them  in  the 
large  fish-kettle  which  it  is  useful  to  have  in  a  bacteriological 
laboratory  for  such  a  purpose.  It  is  preferable  to  have  the 
cages  opening  from  above.  Otherwise  material  which  may  be 
infective  may  be  scratched  out  of  the  cage  by  the  animal.  The 
general  condition  of  the  animal  is  to  be  observed,  how  far  it 
differs  from  the  normal,  whether  there  is  increased  rapidity  of 
breathing,  etc.  The  temperature  is  usually  to  be  taken.  This 
is  generally  done  per  rectum.  The  thermometer  (the  ordinary 
clinical  variety)  is  smeared  with  vaselin,  and  the  bulb  inserted 
just  within  the  sphincter,  where  it  is  allowed  to  remain  for  a 
minute ;  it  is  then  pushed  well  into  the  rectum  for  five  minutes. 
If  this  precaution  be  not  adopted  a  reflex  contraction  of  the 
vessels  may  take  place,  which  is  likely  to  vitiate  the  result  by 
giving  too  low  a  reading. 

Collodion  Capsules. — These  have  been  used  to  allow  the 
sojourn  of  bacteria  within  the  animal  body  without  their  coming 
into  contact  with  the  cells  of  the  tissues.  Various  substances 
in  solution  can  pass  in  either  direction  through  the  wall  by 
diffusion,  but  the  wall  is  impermeable  alike  to  bacteria  and 
leucocytes.  The  following  method  of  preparing  such  capsules  is 
that  of  M'Rae  modified  by  Harris  : — A  gelatin  capsule,  such  as 
is  used  by  veterinary  surgeons,  is  taken,  and  in  one  end  there 
is  fixed  a  small  piece  of  thin  glass  tubing  by  gently  heating  the 
glass  and  inserting  it.  The  tube  becomes  fixed  when  quite  cold, 
and  the  junction  is  then  painted  round  with  collodion,  which 
is  allowed  to  dry  thoroughly.  The  bore  of  the  tubing  is  cleared 
of  any  obstructing  gelatin,  and  the  whole  capsule  is  dipped  into 
a  solution  of  collodion  so  as  to  coat  it  completely.  The  collodion 
is  allowed  to  dry,  and  the  coating  is  repeated ;  it  is  also  advis- 
able to  «j  strengthen  the  layer  by  further  painting  it  at  the 
extremity  and  at  the  junction.  The  interior  of  the  capsule  is 


AUTOPSIES  ON  ANIMALS  145 

then  filled  with  water  by  a  fine  capillary  pipette,  and  the  capsule 
is  placed  in  hot  water  in  order  to  liquefy  the  gelatin,  which 
can  be  removed  from  the  interior  by  means  of  the  fine  pipette. 
The  sac  is  filled  with  bouillon  and  is  placed  in  a  tube  of 
bouillon.  It  is  then  sterilised  in  the  autoclave.  A  small 
quantity  of  the  bouillon  is  removed,  and  the  contents  are 
inoculated  with  the  particular  bacterium  to  be  studied,  or  an 
emulsion  of  the  bacterium  is  added.  The  glass  tubing  is  seized 
in  sterile  forceps,  and  is  sealed  off  in  a  small  flame  a  short 
distance  above  the  junction.  The  closed  sac  ought  then  to  be 
placed  in  a  tube  of  sterile  bouillon  to  test  its  impermeability. 
The  result  is  satisfactory  if  no  growth  occurs  in  the  surrounding 
medium.  The  sac  with  its  contents  can  now  be  transferred  to 
the  peritoneal  cavity  of  an  animal. 

Autopsies  on  Animals  dead  or  killed  after  Inoculation.— 
These  should  be  made  as  soon  as  possible  after  death — in  fact, 
it  is  preferable  to  kill  the  animal  when  it  shows  serious  signs  of 
illness.  It  is  necessary  to  have  some  shallow  troughs,  con- 
structed either  of  metal  or  of  wood  covered  with  metal,  conveni- 
ently with  sheet  lead,  and  having  a  perforation  at  each  corner 
to  admit  a  tape  or  strong  cord.  The  animal  is  tightly  stretched 
out  in  the  trough  and  tied  in  position.  The  size  of  the  trough 
will  therefore  have  to  vary  with  the  size  of  the  outstretched 
body  of  the  animal  to  be  examined.  In  certain  cases  it  is  well 
to  soak  the  surface  of  the  animal  in  carbolic  acid  solution 
(1  to  20)  or  in  corrosive  sublimate  (1  to  1000)  before  it  is  tied 
out.  This  not  only  to  a  certain  extent  disinfects  the  skin,  but, 
what  is  more  important,  prevents  hairs  which  might  be  affected 
with  pathogenic  products  from  getting  into  the  air  of  the 
laboratory.  The  instruments  necessary  are  scalpels  (preferably 
with  metal  handles),  dissecting  forceps,  and  scissors.  They  are 
to  be  sterilised  by  boiling  for  five  minutes.  This  is  conveniently 
done  in  one  of  the  small  portable  sterilisers  used  by  surgeons. 
Two  sets  at  least  ought  to  be  used  in  an  autopsy,  and  they  may 
l»f  placed,  after  boiling,  on  a  sterile  glass  plate  covered  by  a 
bell-jar.  It  is  also  necessary  to  have  a  medium-sized  hatchet- 
si  uiped  cautery,  or  other  similar  piece  of  metal.  It  is  well  to 
have  prepared  a  few  freshly- drawn-out  capillary  tubes  stored  in 
a  sterile  cylindrical  glass  vessel,  and  also  some  larger  sterile  glass 
pipettes.  The  hair  of  the  abdomen  of  the  animal  is  removed. 
If  some  of  the  peritoneal  fluid  is  wanted,  a  band  should  be 
cauterised  down  the  linea  alba  from  the  sternum  to  the  pubes, 
and  another  at  right  angles  to  the  upper  end  of  this ;  an  incision 
should  be  made  in  the  middle  of  these  bands,  and  the  abdominal 
10 


146  INOCULATION  OF  ANIMALS 

walls  thrown  to  each  side.  One  or  more  capillary  tubes  should 
then  be  filled  with  the  fluid  collected  in  the  flanks,  the  fluid 
being  allowed  to  run  up  the  tube  and  the  point  sealed  off;  or  a 
larger  quantity,  if  desired,  is  taken  in  a  sterile  pipette.  If 
peritoneal  fluid  be  not  wanted,  then  an  incision  may  be  made 
from  the  episternum  to  the  pubes,  and  the  thorax  and  abdomen 
opened  in  the  usual  way.  The  organs  ought  to  be  removed  with 
another  set  of  instruments,  and  it  is  convenient  to  place  them 
pending  examination  in  deep  Petri's  capsules  (sterile).  It  is 
generally  advisable  to  make  cultures  and  film  preparations  from 
the  heart's  blood.  To  do  this,  open  the  pericardium,  sear  the 
front  of  the  right  ventricle  with  a  cautery,  make  an  incision  in 
the  middle  of  the  part  seared,  and  remove  some  of  the  blood 
with  a  capillary  tube  for  future  examination ;  or,  introducing  a 
platinum  eyelet,  inoculate  tubes  and  make  cover-glass  prepara- 
tions at  once.  To  examine  any  organ,  sear  the  surface  with  a 
cautery,  cut  into  it,  and  inoculate  tubes  and  make  film  prepara- 
tions with  a  platinum  loop.  For  removing  small  parts  of  organs 
for  making  inoculations  on  tubes,  a  small  platinum  spud  is  very 
useful,  as  the  ordinary  wires  are  apt  to  become  bent.  Place 
pieces  of  the  organs  in  some  preservative  fluid  for  miscroscopic 
examination.  The  organs  ought  not  to  be  touched  with  the 
fingers.  When  the  examination  is  concluded,  the  body  should 
have  corrosive  sublimate  or  carbolic  acid  solution  poured  over  it, 
and  be  forthwith  burned.  The  dissecting  trough  and  all  the 
instruments  ought  to  be  boiled  for  half  an  hour.  The  amount 
of  precaution  to  be  taken  will,  of  course,  depend  on  the  character 
of  the  bacterium  under  investigation,  but  as  a  general  rule  every 
care  should  be  used. 


CHAPTER   V. 

BACTERIA  IN  AIR,  SOIL,  AND  WATER. 
ANTISEPTICS. 

IT  is  impossible  here  to  do  more  than  indicate  the  chief  methods 
which  are  employed  by  bacteriologists  in  the  investigation  of  the 
bacteria  present  in  air,  soil,  and  water,  and  to  add  an  outline  of 
the  chief  results  obtained.  In  dealing  with  the  latter,  the  subject 
lias  been  approached  mainly  from  the  standpoint  of  the  bearings 
which  the  results  have  towards  human  pathology.  In  dealing 
with  antiseptics,  so  far  as  possible  the  effects  of  the  various 
agents  on  the  chief  pathogenic  bacteria  have  been  given,  though 
in  many  cases  our  information  is  very  imperfect. 

AIR. 

Very  little  information  of  value  can  be  obtained  from  the 
examination  of  the  air,  but  the  following  are  the  chief  methods 
used,  along  with  the  results  obtained.  More  can  be  learned 
from  the  examination  of  atmospheres  experimentally  contamin- 
ated than  by  the  investigation  of  the  air  as  it  exists  under 
natural  conditions. 

Methods  of  Examination. — The  methods  employed  vary  with  the 
objects  in  view.  If  it  be  sought  to  compare  the  relative  richness  of 
different  atmospheres  in  organisms,  and  if  the  atmospheres  in  question 
be  fairly  quiescent,  then  it  is  sufficient  to  expose  gelatin  plates  for 
definite  times  in  the  rooms  to  be  examined.  Bacteria,  or  the  particles  of 
dust  carrying  them,  fall  on  the  plates,  and  from  the  number  of  colonies 
which  develop  a  rough  idea  of  the  richness  of  the  air  in  bacteria  can  be 
obtained.  Petri  states  that  in  five  minutes  the  bacteria  present  in 
10  litres  of  air  are  deposited  on  100  square  centimetres  of  a  gelatin  plate. 

More  complete  results  are  available  when  some  method  is  employed  by 
which  the  bacteria  in  a  given  quantity  of  air  are  examined.  The  oldest 
in.'tlin  1  employed,  and  one  which  is  still  used,  is  that  of  Hesse.  The 
apparatus  is  shown  in  Fig.  48.  It  consists  of  a  cylindrical  tube  a  about 
20  inches  long  and  2  inches  in  diameter.  At  one  end  this  is  closed  by  a 
rubber  cork  having  a  piece  of  quill  tubing,  /,  passing  through  it  and 


148 


BACTERIA  IN  AIR 


projecting  some  distance  into  the  interior.  For  use  the  tube  is  sterilised 
in  a  tall  "Koch,"  and  then  a  quantity  of  peptone  gelatin,  sufficient  to 
cover  the  whole  interior  to  the  thickness  of  an  ordinary  gelatin  plate,  is 
poured  in.  This  gelatin  is  kept  from  escaping  by  the  projection  of  the 
quill  tubing  into  the  lumen  of  the  large  tube.  A  plug  of  cotton  wool  is 
now  placed  in  the  outer  end  of  the  quill  tubing.  Over  the  other  end  of 
the  large  tube  is  tied  a  sheet  of  rubber  having  a  hole  about  a  quarter  of 
an  inch  in  diameter  in  its  centre,  and  over  this  again  is  tied  a  piece 
of  similar  but  unperforated  sheet  rubber.  The  tube  is  then  sterilised 

in  the  tall  "Koch."  On 
removal  from  this  it  is 
rolled,  after  the  manner 
of  an  Esmarch's  tube 
(q.v.),  till  the  gelatin  is 
set  as  a  layer  over  its 
interior,  and  it  is  then 
placed  horizontally  on  the 
tripod  as  shown.  The 
other  part  of  the  appa- 
ratus is  an  aspirator,  by 
means  of  which  a  known 
quantity  of  air  can  be 
brought  in  contact  with 
the  gelatin.  It  consists 
of  two  conical  glass  flasks 
connected  by  means  of  a 
tube  which  passes  through 
the  cork  of  each  down  to 
the  bottom  of  the  flask. 
When  this  tube  is  filled 
with  water  it,  of  course, 
can  act  as  a  syphon  tube 
between  volumes  of  water 
in  the  flasks.  Such  a 
syphon  system  being  es- 
tablished, the  levels  of 
the  water  are  marked  on 
the  flasks,  and  to  one  a 
litre  of  water  is  added  ; 
by  depressing  flask  b  the 
whole  litre  can  be  got 
into  it,  and  the  connect- 
ing tube  c  is  then  clamped.  The  two  flasks  are  now  connected  by  a 
rubber  tube  with  the  tube/,  the  clamp  on  c  is  opened,  and  the  passing  of 
a  litre  of  water  into  d  will  draw  a  litre  of  air  through  the  gelatin  tube, 
when  the  outer  rubber  sheet  is  removed  from  the  end  and  the  clamp  h 
opened.  By  disconnecting  at  g  and  reversing  the  syphon  flasks,  another 
litre  can  be  sucked  through,  and  so  any  desired  quantity  of  air  can  be 
brought  in  contact  with  the  gelatin.  The  speed  ought  not  to  be  more 
than  one  litre  in  two  minutes,  and  in  such  a  case  practically  all  the 
organisms  will  be  found  to  have  fallen  out  of  the  air  on  to  the  gelatin 
in  the  course  of  their  transit.  This  fact  can  be  tested  by  interposing 
between  the  tube  a  and  the  aspirator  a  second  tube  prepared  in  the  same 
way,  which  ought,  of  course,  to  show  no  growth.  When  forty-eight 
hours  at  20°  C.  or  four  days  at  lower  temperature  have  elapsed,  the 


FIG.  48. — Hesse's  tube,  mounted  for  use. 


PETRT'S  SAND-FILTER  METHOD 


149 


i  which  develop  in  a  may  be  counted.  The  disadvantage  of  the 
method  is  that  if  particles  of  dust  carrying  more  than  one  bacterium 
alight  on  the  gelatin,  these  bacteria  develop  in  one  colony,  and  thus  the 
numeration  results  may  be  too  low  ;  difficulties  may  also  arise  from 
liquefying  colonies  developing  in  the  upper  parts  of  the  tube  and  running 
over  the  gelatin. 

Petri's  Sand-Filter  Method.— A  glass  tube  open  at  both  ends,  and 
about  3i  inches  long  and  half  an  inch  wide,  is  taken,  and  in  its  centre  is 
placed  a  transverse  diaphragm  of  very  fine  iron  gauze 
(Fig.  49,  e.} ;  on  each  side  of  this  is  placed  some  fine 
quartz  sand  which  has  been  well  washed,  dried,  and 
burned  to  remove  all  impurities,  and  this  is  kept  in 
position  by  cotton  plugs.  The  whole  is  sterilised  by 
dry  heat.  One  plug  is  removed,  and  a  sterile  rubber 
cork,  r,  inserted,  through  which  a  tube,  d,  passes  to 
an  exhausting  apparatus.  The  tube  is  then  clamped 
in  an  upright  position  in  the  atmosphere  to  be  ex- 
amined, with  the  remaining  plug,  /,  uppermost. 
The  latter  is  removed  and  the  air  sucked  through. 
Difficulty  may  be  experienced  from  the  resistance  of 
the  sand  if  quick  filtration  be  attempted.  The  best 
means  to  adopt  is  to  use  an  air-pump — the  amount 
of  air  drawn  per  stroke  of  which  is  accurately  known 
— and  to  have  a  manometer  (as  in  Fig.  30)  interposed 
between  the  tube  and  the  pump.  Between  each  two 
strokes  of  the  air-pump  the  mercury  is  allowed  to 
return  to  zero.  After  the  required  amount  of  air  has 
passed,  the  sand  a  is  removed,  and  is  distributed 
among  a  number  of  sterile  gelatin  tubes  which  arc 
well  shaken  ;  plate  cultures  are  then  made,  and 
when  growth  has  occurred  the  colonies  are  enumer- 
ated ;  the  sand  b  is  similarly  treated,  and  acts  as  a 
control. 

When  it  is  necessary  to  examine  air  for  particular 
organisms,  special  methods  must  often   be  adopted. 
Thus  in  the  case  of  the  suspected  presence  of  tubercle  bacilli  a  given 
quantity  of  air  is  drawn  through  a  small  quantity  of  water  and  then 
injected  into  a  guinea-pig. 


d 


FIG.  49.— Petri's 
sand  filter. 


It  must  be  admitted  that  comparatively  little  information 
bearing  on  the  harmlessness  or  harmfulness  of  the  air  is  obtain- 
able by  the  mere  enumeration  of  the  living  organisms  present, 
for  under  certain  conditions  the  number  may  be  increased  by 
the  presence  of  many  individuals  of  a  purely  non-pathogenic 
character.  The  organisms  found  in  the  air  belong  to  two 
groups — firstly,  a  great  variety  of  bacteria  ;  secondly,  yeasts  and 
the  spores  of  moulds  and  of  the  lower  fungi.  With  regard  to 
the  spores,  the  organisms  from  which  they  are  derived  often 
consist  of  felted  masses  of  threads,  from  which  are  thrust  into 
the  air  special  filaments,  and  in  connection  with  these  the  spores 
are  formed.  By  currents  of  air  these  latter  can  easily  be  detached. 


150  BACTERIA  IN  AIR 

and  may  float  about  in  a  free  condition.  With  the  bacteria,  on 
the  other  hand,  the  case  is  different.  Usually  these  are  growing 
together  in  little  masses  on  organic  materials,  or  in  fluids,  and 
it  is  very  much  by  the  detachment  of  minute  particles  of  the 
substratum  that  the  organisms  become  free.  The  entrance  of 
bacteria  into  the  air,  therefore,  is  associated  with  conditions 
which  favour  the  presence  of  dust,  minute  droplets  of  fluid,  etc. 
The  presence  of  dust,  in  particular,  would  specially  favour  a  large 
number  of  bacteria  being  observed,  and  this  is  the  case  with  the 
air  in  many  industrial  conditions,  where  the  bacteria,  though 
numerous,  may  be  quite  innocuous.  Great  numbers  of  bacteria 
thus  may  not  indicate  any  condition  likely  to  injure  health,  and 
this  may  be  true  also  even  when  the  bacteria  come  from  the 
crowding  together  of  a  number  of  healthy  human  beings.  On 
the  other  hand,  there  is  no  doubt  that  disease  germs  can  be 
disseminated  by  means  of  the  air.  The  possibility  of  this 
has  been  shown  experimentally  by  infecting  the  mouth  with  the 
b.  prodigiosus,  which  is  easily  recognised  by  its  brilliantly 
coloured  colonies,  and  then  studying  its  subsequent  distribution. 
Most  important  here  is  the  infection  of  the  air  from  sick  persons. 
The  actions  of  coughing,  sneezing,  speaking,  and  even  of  deep 
breathing,  distribute,  often  to  a  considerable  distance,  minute 
droplets  of  secretions  from  the  mouth,  throat,  and  nose,  and  these 
may  float  in  the  air  for  a  considerable  time.  Even  live  hours 
after  an  atmosphere  has  been  thus  infected  evidence  may  be 
found  of  bacteria  still  floating  free.  Before  this  time,  however, 
most  of  the  bacteria  have  settled  upon  various  objects,  where 
they  rapidly  dry,  and  are  no  longer  displaceable  by  ordinary  air 
currents.  The  diseases  of  known  etiology  where  infection  can 
thus  take  place  are  diphtheria,  influenza,  pneumonia,  and  phthisis  ; 
and  here  also  possibly  typhus  fever  and  measles  are  to  be 
added,  though  the  morbific  agents  are  unknown.  In  the  case  of 
phthisis,  the  deposition  of  tubercle  bacilli  has  been  demonstrated 
on  cover-glasses  held  before  the  mouths  of  patients  while  talking, 
and  animals  made  to  breathe  directly  in  front  of  the  mouths  of 
such  patients  have  become  infected  with  tuberculosis.  Apart 
from  direct  infection  from  individuals,  however,  pathogenic 
bacteria  may  be  spread  in  some  cases  from  the  splashing  of 
infected  water,  as  from  a  sewage  outfall.  This  possibility  has  to 
be  recognised  especially  in  the  cases  of  typhoid  and  cholera. 
Besides  infection  through  fluid  particles,  infection  can  be  caused 
in  the  air  by  dust  coming  from  infected  skin  or  clothes, 
etc.  Fliigge,  in  dealing  with  this  subject  in  an  experimental 
inquiry,  distinguishes  between  large  particles  of  dust  which 


DISTRIBUTION  OF  BACTERIA  BY  AIR         151 

nM|iihv  ;ui  air  current  moving  at  the  rate  of  1  centimetre  per 
second  in  l«-rp  them  suspended,  and  the  finer  dust  which  can  In- 
kept  in  suspension  by  currents  moving  at  from  1  to  4  milli- 
metres per  second.  In  the  former  case,  when  once  the  particles 
settle  they  cannot  be  displaced  by  currents  of  air  except  when 
these  are  moving  at,  at  least,  5  metres  per  second,  but  the 
brushing,  shaking,  or  beating  of  objects  may,  of  course,  distribute 
tin-in.  In  the  case  of  the  liner  dust  the  particles  will  remain  for 
long  suspended,  and  when  they  have  settled  can '  be  more  easily 
displaced,  as  by  the  waving  of  an  arm,  breathing,  etc.  With  re- 
gard to  infection  by  dust,  a  most  important  factor,  howrever,  is 
whether  or  not  the  infecting  agent  can  preserve  its  vitality  in 
a  dry  condition.  In  the  case  of  a  sporing  organism  such  as 
anthrax,  vitality  is  preserved  for  long  periods  of  time,  and  great 
resistance  to  drying  is  also  possessed  by  the  tubercle  and 
diphtheria  bacilli ;  but  apart  from  such  cases  there  is  little 
doubt  that  infection  is  usually  necessarily  associated  with  the 
transport  of  moist  particles,  and  is  thus  confined  to  a  limited 
area  around  a  sick  person.  Among  diseases  which  may  occasion- 
ally be  thus  spread,  cholera  and  typhoid  have  been  classed. 
Considerable  controversy  has  arisen  with  regard  to  certain  out- 
breaks of  the  latter  disease,  which  have  apparently  been  spread 
by  dusty  winds,  although  we  have  the  fact  that  the  typhoid 
bacillus  does  not  survive  being  dried  even  for  a  short  time. 
It  appears,  however,  that  in  such  epidemics  the  transport  of 
infection  by  means  of  insects  carried  by  the  wind  has  not  been 
entirely  excluded. 

As  in  the  cases  of  the  soil  and  of  water,  presently  to  be  described, 
attempts  have  been  made  to  obtain  indirect  evidence  of  the  contamination 
ni  the  air  from  human  sources.  Thus  Gordon  has  shown  that  certain 
streptococci  are  common  in  the  saliva  ;  these  usually  correspond  to  the 
f'ococcus  salirarius  of  Andrewes  »«d  Horder  (q.v.)  in  that  they  grow 
at  37°  C.,  form  acid  and  clot  in  litmus  milk,  reduce  neutral-red,  and  fer- 
ment saccharose,  lactose,  and  raffinose.  Andrewes  and  Horder  also  describe 
another  group, — sir.  cquinm, — as  common  in  London  air,  which  they 
think  is  there  derived  from  horse  dung.  Thus  the  finding  of  streptococci 
of  the  first  group  in  plates  exposed  to  air  would  indicate  that  a  human 
source  was  probable,  and,  if  the  observation  were  made  on  air  from  the 
neighbourhood  of  a  sick  person,  that  risk  of  the  dissemination  of  disease 
g«Tins  was  present.  The  value  of  this  as  a  practical  method  has  yet  to  he 
determined, 

Son.. 

The  investigation  of  the  bacteria  which  may  be  found  in  the 
soil  is  undertaken  from  various  points  of   view.     Information 


152  BACTERIA  IN  SOIL 

may  be  desired  as  to  the  change  its  composition  undergoes  by 
a  bacterial  action,  the  result  of  which  may  be  an  increase 
in  fertility  and  thus  in  economic  value.  Under  this  head  may 
be  grouped  inquiries  relating  to  the  bacteria  which  convert 
ammonia  and  its  salts  into  nitrates  and  nitrites,  and  to  the 
organisms  concerned  in  the  fixation  of  the  free  nitrogen  of  the 
air.  The  discussion  of  the  questions  involved  in  such  inquiries 
is  outside  the  scope  of  the  present  chapter,  which  is  more  con- 
cerned with  the 'relation  of  the  bacteriology  of  the  soil  to  questions 
of  public  health.  So  far  as  this  narrower  view  is  concerned,  soil 
bacteria  are  chiefly  of  importance  in  so  far  as  they  can  be  washed 
out  of  the  soils  into  potable  water  supplies.  An  important  aspect 
of  this  question  thus  is  as  to  the  significance  of  certain  bacterio- 
logical appearances  in  a  water  in  relation  to  the  soil  from  which 
it  has  come  or  over  which  it  has  flowed.  In  this  country  these 
questions  have  been  chiefly  investigated  by  Houston,  and  it  is 
from  his  papers  that  the  following  account  is  largely  taken. 

Methods  of  Examination. — For  examination  of  soil  on  surface  or  not 
far  from  surface,  Houston  recommends  tin  troughs  10  in.  by  3  in.,  and 
pointed  at  one  extremity,  to  be  wrapped  in  layers  of  paper  and  sterilised 
by  dry  heat.  If  several  of  these  be  provided,  then  the  soil  can  be  well 
rubbed  up  and  a  sample  secured  and  placed  in  a  sterile  test-tube  for 
examination  as  soon  as  convenient  after  collection.  If  samples  are  to 
be  taken  at  some  depth  beneath  the  surface,  then  a  special  instrument 
of  which  many  varieties  have  been  devised  must  be  used.  The  general 
form  of  these  is  that  of  a  gigantic  gimlet  stoutly  made  of  steel.  Just 
above  the  point  of  the  instrument  the  shaft  has  in  it  a  hollow  chamber, 
and  a  sliding  lateral  door  in  this  can  be  opened  and  shut  by  a  mechanism 
controlled  at  the  handle.  The  chamber  being  sterilised  and  closed,  the 
instrument  is  bored  to  the  required  depth,  the  door  is  slid  back,  and  by 
varying  devices  it  is  effected  that  the  chamber  is  filled  with  earth  ;  the 
door  is  reclosed  and  the  instrument  withdrawn. 

In  any  soil  the  two  important  lines  of  inquiry  are  first,  as  to  the  total 
number  of  organisms  (usually  reckoned  per  gramme  of  the  fresh  sample) ; 
and  secondly,  as  to  the  varieties  of  organisms  present.  The  number  of 
organisms  present  in  a  soil  is  often,  however,  so  enormous  that  it  is  con- 
venient to  submit  only  a  fraction  of  a  gramme  to  examination.  The 
method  employed  is  to  weigh  the  tube  containing  the  soil,  shake  out  an 
amount  of  about  the  size  of  a  bean  into  a  litre  of  distilled  water,  and 
revveigh  the  tube.  The  amount  placed  in  the  water  is  distributed  as 
thoroughly  as  possible  by  shaking,  and,  if  necessary,  by  rubbing  down 
with  a  sterile  glass  rod,  and  small  quantities  .measured  from  a  graduated 
pipette  are  used  for  the  investigation.  For  estimating  the  total  number  of 
organisms  present  in  the  portion  of  soil  used,  small  quantities,  say  "1  c.c. 
and  1  c.c.,  of  the  fluid  are  added  to  melted  tubes  of  ordinary  alkaline 
peptone  gelatin  ;  after  being  shaken,  the  gelatin  is  plated,  incubated  at 
22°  C.,  and  the  colonies  are  counted  as  late  as  the  liquefaction,  which 
always  occurs  round  some  of  them,  will  allow.  From  these  numbers  the 
total  number  of  organisms,  which  grow  in  gelatin,  in  a  given  amount  of 
soil  can  be  calculated. 


BACTERIA  IN  SOIL  153 

The  numbers  of  bacteria  in  the  soil  vary  very  much.  Accord- 
ing to  Houston's  results,  fewest  occur  in  uncultivated  sandy  soils, 
these  containing  on  an  average  100,000  per  gramme.  Peaty  soils, 
though  rich  in  organic  matter,  also  give  low  results,  it  being 
possible  that  the  acidity  of  such  soils  inhibits  free  bacterial 
growth.  Garden  soils  yield  usually  about  1,500,000  bacteria 
per  gramme,  but  the  greatest  numbers  are  found  in  soils  which 
have  been  polluted  by  sewage,  when  the  figures  may  rise  to 
several  millions.  In  addition  to  the  enumeration  of  the  numbers 
of  bacteria  present,  it  is  a  question  whether  something  may  not 
be  gained  from  a  knowledge  of  the  number  of  spores  present  in 
a  soil  relative  to  the  total  number  of  bacteria.  This  is  a  point 
which  demands  further  inquiry,  especially  by  the  periodic  investi- 
gation of  examples  of  different  classes  of  soils.  The  method  is  to 
take  1  c.c.  of  such  a  soil  emulsion  as  that  just  described,  add  it 
to  10  c.c.  of  gelatin,  heat  for  ten  minutes  at  80°  C.  to  destroy 
the  non-spored  bacteria,  plate,  incubate,  and  count  as  before. 

Besides  the  enumeration  of  the  numbers  of  bacteria  present  in 
a  soil,  an  important  question  in  its  bacteriological  examination 
lies  in  inquiring  what  kinds  of  bacteria  are  present  in  any  par- 
ticular case.  Practically  this  resolves  itself  into  studying  the 
most  common  bacteria  present,  for  the  complete  examination  of 
the  bacterial  flora  of  any  one  sample  would  occupy  far  too  much 
time.  Of  these  common  bacteria  the  most  important  are  those 
from  whose  presence  indications  can  be  gathered  of  the  con- 
tamination of  the  soil  by  sewage,  for  from  the  public  health 
standpoint  this  is  by  far  the  most  important  question  on  which 
bacteriology  can  shed  light. 

Bacillus  tnycoides. — This  bacillus  is  1'6  to2'4  /u.  in  length,  and  about 
•9  JJL  in  breadth.  It  grows  in  long  threads  which  often  show  motility. 
It  can  be  readily  stained  by  such  a  combination  as  carbol-thionin,  and 
retains  the  dye  in  Gram's  method.  All  ordinary  media  will  support  its 
growth,  and,  in  surface  growths  on  agar  or  potato,  spore  formation  is 
readily  produced.  Its  optimum  temperature  is  about  18°  C.  On  gelatin 
plates  it  shows  a  very  characteristic  appearance.  At  first  under  a  low 
power  it  shows  a  felted  mass  of  filaments  throwing  out  irregular  shoots 
from  the  centre,  and  later  to  the  naked  eye  these  appear  to  be  in  the 
form  of  thick  threads  like  the  growth  of  a  mould.  They  rapidly  spread 
over  the  surface  of  the  medium,  and  the  whole  resembles  a  piece  of  wet 
teased-out  cotton  wool.  The  gelatin  is  liquefied. 

Cladothrices. — Of  these  several  kinds  are  common  in  the  soil.  The 
ordinary  cladothrix  dichotoma,  is  among  them.  This  organism  appears 
as  a  colourless  flocculent  growth  with  an  opaque  centre,  and  can  be  seen 
under  the  microscope  to  send  out  into  the  medium  apparently  branched 
threads  which  vary  in  thickness,  being  sometimes  2  /*  across.  They 
consist  of  rods  enclosed  in  a  sheath.  These  rods  may  divide  at  any 
point,  and  thus  the  terminal  elements  may  be  pushed  along  the  sheath. 


154  BACTERIA  IN  SOIL 

Sometimes  the  sheath  ruptures,  and  thus  by  the  extrusion  of  these 
dividing  .cells  and  their  further  division  the  branching  appearance  is 
originated.  Reproduction  takes  place  by  the  formation  ot'gonidia  in  the 
interior  of  the  terminal  cells.  These  gonidia  acquire  at  one  end  a  bundle 
of  flagella,  and  for  some  time  swim  free  before  becoming  attached  and 
forming  a  new  colony.  Houston  describes  as  occurring  in  the  soil  another 
variety,  which  with  similar  microscopic  characters  appears  as  a  brownish 
growth  with  a  pitted  surface  and  diffuses  a  Bismarck-brown  pigment 
into  the  gelatin  which  it  liquefies. 

A  few  experiments  made  with  an  ordinary  field  soil  will,  however, 
familiarise  the  worker  with  the  non-pathogenic  bacteria  usually  present. 
We  have  referred  to  these  two  because  of  their  importance.  In  regard 
to  pathogenic  organisms,  especially  in  relation  to  possible  sewage  con- 
tamination, attention  is  to  be  directed  to  three  groups  of  organisms, 
those  resembling  the  b.  coli,  the  bacillus  enteritidis  sporogenes,  and  the 
streptococcus  pyogenes.  The  characters  of  the  first  two  of  these  will 
be  found  in  the  chapter  on  Typhoid  Fever  ;  of  the  third  in  Chapter  VII. 
For  the  detection  of  these  bacteria  Houston  recommends  the  following 
procedure  : — 

(a)  The  B.   coli  Group. — A  third    of  a  gramme  of  soil   is  added   to 
10  c.c.   broth  containing  '2  per  cent,  of  phenol  and  incubated  at  37°  C. 
In  this  medium  very  few  if  any  other  bacteria  except  those  of  the  b.  coli 
group  will  grow,  so  that  if  after  twenty-four  hours  a  turbidit}r  appears, 
some   of  the  latter  may  be  suspected  to  be  present.     In  such  a  case  a 
loopful  of  the  broth  is  shaken  up  in  5  c.c.  sterile  distilled  water,  and  of 
this  one  or  two  loopfuls  are  spread  over  the  surface  of  a  solid  plate  of 
phenol  gelatin  in  a   Petri  capsule  either  by  means  of  the  loop  or  of  a 
small   platinum   spatula,    and   the    plate   is   incubated   at   20°  C.     Any 
colonies  which  resemble  b.  coli  are  then  examined  by  the  culture  methods 
detailed  under  that  organism.     Further,  all  organisms  having  the  micro- 
scopic appearances  of  b.  coli,  and  which  generally  conform  to  its  culture 
reactions,  are  to  be  reckoned  in  the  coli  group.     The  media  of  MacConkey 
and  Drigalski  are  very  useful  in  connection  with  the  plating  and  separa- 
tion of  such  soil  organisms  (vide  pp.  50,  47). 

(b)  The   Bacillus  enleritidis  sporogenes. — To  search  for  this  organism 
1  gramme  of  the  soil  is  thoroughly  distributed  in  100  c.c.  sterile  distilled 
water,  and  of  this  1  c.c.,  '1  c.c.,  and  '01  c.c.  is  added  to  each  of  three 
sterile  milk  tubes.     These  are  heated  to  80°  C.  for  ten  minutes,  and  then 
cultivated  anaerobically  at  37°  C.  for  twenty-four  hours.     If  the  charac- 
teristic appearances  seen  in  such  cultures  of  the  b.   enteritidis  (q.v.}  are 
developed,  then  it  may  fairly  safely  be  deduced  that  it  is  this  organism 
which  has  produced  them. 

(c)  FcKcal  Streptococci. — The  method  here  is  to  pour  out  a  tube  of  agar 
into  a  Petri  capsule,  and  when  it  has  solidified  to  spread  out  *1  c.c.   of 
the  emulsion  of  soil  over  it  and  incubate  at  37°  C.  for  twenty-four  hours. 
At  this  temperature  many  of  the   non-pathogenic   bacteria   grow  with 
difficulty,  and  thus  the  number  of  colonies  which  develop  is  relatively 
small.     Colonies  having  appearances  resembling  those  of  the  streptococcus 
pyogenes  (q.v.}  can  thus  be  investigated. 

Another  method  is  that  of  Prescott  and  Winslow  modified  by  Mair. 
This  depends  on  the  fact  that  when  b.  coli  and  streptococci  are  growing 
together  in  glucose  broth,  as  the  medium  becomes  acid  the  streptococci 
tend  to  outgrow  the  b.  coli.  If  lactose  agar  plates  be  made  at  this  stage, 
the  colonies  of  streptococci,  being  small  and  intensely  red,  can  be  distin- 
guished from  the  larger  and  less  acid  colonies  of  the  b.  coli.  They  can 


BACTERIA  IN  SOIL  155 

then  be  picked  off  for  investigation.  It  is  evident  lli.it  here  tlie  method 
must  be  adopted  of  taking  as  a  measure  of  the  number  of  streptococci 
present  the  least  quantity  of  the  original  fluid  in  which  evidence  of  their 

presence  can  be  detected. 

\\V  may  now  ^ive  in  brief  the  results  at  which  Houston  has 
arrived  by  the  application  of  these  methods.  First  of  all,  un- 
cultivated soils  contain  very  few,  if  any,  representatives  of  the 
b.  mycoides,  and  this  is  also  true  to  a  less  extent  of  the 
dadothrices.  Cultivated  soils,  on  the  other  hand,  do  practically 
always  contain  thrsr  organisms.  With  regard  to  the  b.  coli, 
its  presence  in  a  soil  must  be  looked  on  as  indicative  of 
recent  pollution  with  excremental  matter.  The  presence  of 
b.  eiiti'ritidis  is  also  evidence  of  such  pollution,  but  from  the  fact 
that  this  is  a  sporing  organism  the  pollution  may  not  have  been 
recent.  With  regard  to  the  streptococci,  on  the  other  hand,  the 
opinion  is  advanced  that  their  presence  is,  on  account  of  their 
tWble  viability  outside  the  animal  body,  to  be  looked  on  as 
evidence  of  extremely  recent  excremental  |x)llution.  The  very- 
great  importance  of  these  results  in  relation  to  the  bacterio- 
logical examination  of  water  supplies  will  be  at  once  apparent, 
and  will  be  referred  to  again  in  connection  with  this  subject. 

While  such  means  have  been  advanced  for  the  obtaining  of 
indirect  evidence  of  excremental  pollution  of  soil,  and  therefore 
of  a  pollution  dangerous  to  health  from  the  possible  presence  of 
pathogenic  organisms  in  excreta,  investigations  have  also  been 
conducted  with  regard  to  the  viability  in  the  soil  of  pathogenic 
bacteria,  especially  of  those  likely  to  be  present  in  excreta,  namely, 
the  typhoid  and  cholera  organisms.  The  solution  of  this  problem 
is  attended  with  difficulty,  as  it  is  not  easy  to  identify  these 
•  ir^anisms  when  they  are  present  in  such  bacterial  mixtures  as 
naturally  occur  in  the  soil.  Now  there  is  evidence  that  bacteria 
when  growing  together  often  influence  each  other's  growth  in  an 
unfavourable  wray,  so  that  it  is  only  by  studying  the  organisms  in 
<iuestion  when  growing  in  unsterilised  soils  that  information  can 
l>e  obtained  as  to  what  occurs  in  nature.  For  instance,  it  has 
been  found  that  the  b.  typhosus,  when  grown  in  an  organically 
polluted  soil  which  has  been  sterilised,  can  maintain  its  vitality 
for  fifteen  weeks,  but  if  the  conditions  occurring  naturally  be  so 
far  imitated  by  growing  it  in  soil  in  the  presence  of  a  pure 
culture  of  a  soil  bacterium,  it  is  found  that  sometimes  the 
typhoid  bacillus,  sometimes  the  soil  bacterium  in  the  course 
of  a  few  weeks,  or  even  in  a  few  days,  disappears.  Further,  the 
character  of  the  soil  exercises  an  important  effect  on  the  results ; 
for  instance,  the  typhoid  bacillus  soon  dies  out  in  a  virgin  sandy 


156  BACTERIA  IN  WATER 

soil,  even  when  it  is  the  only  organism  present.  In  experiments 
made  by  sowing  cultures  of  cholera  and  diphtheria  in  plots  in  a 
field,  it  was  found  that  after,  at  the  longest,  forty  days  they  were 
no  longer  recognisable.  Further,  it  is  a  question  whether 
ordinary  disease  organisms,  even  if  they  remain  alive,  can 
multiply  to  any  great  extent  in  soil  under  natural  conditions. 
If  we  are  dealing  with  a  sporing  organism  such  as  the  b. 
anthracis,  the  capacity  for  remaining  in  a  quiescent  condition  of 
potential  pathogenicity  is,  of  course,  much  greater.  The  most 
important  principle  to  be  deduced  from  these  experiments  is  that 
the  ordinary  conditions  of  soil  rather  tend  to  be  unfavourable 
to  the  continued  existence  of  pathogenic  bacteria,  so  that  by 
natural  processes  soil  tends  to  purify  itself.  It  must,  however, 
be  noted  that  such  an  organism  as  the  typhoid  bacillus  can  exist 
long  enough  in  soil  to  be  a  serious  source  of  danger. 

WATER. 

In  the  bacteriological  examination  of  water  three  lines  of 
inquiry  may  have  to  be  followed.  First,  the  number  of  bacteria 
per  cubic  centimetre  may  be  estimated.  Second,  the  kinds  of 
bacteria  present  may  be  investigated.  Third,  it  may  be  necessary 
to  ask  if  a  particular  organism  is  present,  and,  if  so,  in  what 
number  per  c.c.  it  occurs. 

Methods.  — Collection  of  Samples. — In  all  water  examinations  it  is  pre- 
ferable that  the  primary  culture  media  (i.e.  those  to  which  the  water  is 
actually  to  be  added)  should  be  inoculated  at  the  spot  at  which  the  sample 
is  collected.  When  this  is  not  possible,  the  samples  should  be  packed  in 
sawdust  and  ice  and  the  primary  inoculations  made  as  soon  as  possible. 
Otherwise  the  bacteria  will  multiply,  and  an  erroneous  idea  of  the  number 
present  will  be  obtained.  Immediately  after  collection  a  slight  diminution 
in  numbers  may  be  observed,  but  at  any  rate  after  six  hours  an  increase 
over  the  initial  numbers  is  manifest. 

When  samples  have  to  be  taken  for  transport  to  the  laboratory,  these 
are  best  collected  in  8-ounce,  wide-mouthed,  stoppered  bottles,  which 
are  to  be  sterilised  by  dry  heat  (the  stopper  must  be  sterilised  separately 
from  the  bottle  and  not  inserted  in  the  latter  till  both  are  cold,  otherwise 
it  will  be  so  tightly  held  as  to  make  removal  very  difficult). 

In  the  case  of  water  taken  from  a  house  tap,  the  water  should  be  allowed 
to  run  for  some  time  before  the  sample  is  taken,  as  water  standing  in 
pipes  in  a  house  is  under  very  favourable  conditions  for  multiplication  of 
bacteria  taking  place,  and  if  this  precaution  be  not  adopted  an  altogether 
erroneous  idea  of  the  number  present  may  be  obtained. 

With  river  waters  it  is  best  to  immerse  the  sampling  bottle  and  then 
remove  the  stopper  with  forceps.  Care  must  be  taken  not  to  touch  the 
river  bed,  as  the  vegetable  matter  covering  it  contains  many  organisms. 
When  water  has  to  be  taken  from  below  the  surface  of  a  well  or  lako,  a 
weighted  sample  bottle  must  be  used.  Several  special  bottles  have  been 


BACTERIA  IN  WATER  157 

devised  for  such  a  purpose.  Quite  good  results  are  obtained  by  tying 
two  short  lengths  of  string  to  the  neck  and  stopper  of  an  ordinary  bottle 
respectively,  winding  them  round  the  neck  and  enveloping  in  cotton 
wool  ;  any  required  length  of  string  can  afterwards  be  knotted  on  these. 
A  piece  of  lead  can  be  attached  to  the  bottom  of  the  bottle  by  wires 
passing  round  the  neck.  The  whole  is  then  wrapped  in  paper  and 
sterilised.  For  use  the  bottle  is  carefully  lowered  to  the  required  depth 
by  the  string  attached  to  the  neck,  the  stopper  is  jerked  out,  and  the 
bottle  filled.  If  the  bottle  and  stopper  be  rapidly  jerked  through  the 
topmost  layers,  contamination  with  surface  bacteria  does  not  appear  as  a 
serious  factor. 

Counting  of  Bacteria  in  Water. — This  is  done  by  adding  a  given  quantity 
of  water  to  10  c.c.  of  liquefied  gelatin  or  agar,  plating,  and  counting  the 
colonies  which  develop.  The  amount  of  water  added  depends  on  its 
source,  and  varies  from  '1  c.c.  of  a  water  likely  to  have  a  high  bacterial 
content  to  5  c.c.  of  a  purer  water.  It  is  usual  to  inoculate  both  gelatin 
and  agar  tubes.  The  former,  incubated  at  20°  C.,  gives  an  idea  of  the 
numbers  of  bacteria  present  which  grow  at  summer  heat ;  the  latter, 
incubated  at  37Q  C.,  those  which  grow  at  blood-heat.  As  the  pathogenic 
and  intestinal  bacteria  grow  at  this  temperature,  the  determination  of  the 
numbers  of  blood-heat  bacteria  is  important.  The  counts  on  the  two 
media  usually  differ  as  each  is  favourable  to  the  growth  of  its  own  group 
of  organisms.  With  regard  to  the  summer-heat  bacteria  it  is  important 
to  note  that  gelatine  of  a  slightly  greater  alkalinity  than  that  ordinarily 
prepared — such  an  increased  degree  as  is  caused  by  the  addition  of 
•01  grm.  of  Na2C03  to  10  c.c.  peptone  gelatin — will  give  a  greater  yield 
of  colonies.  In  the  case  of  both  gelatin  and  agar  plates  usually  forty- 
••ight  hours'  incubation  is  allowed  before  the  colonies  are  counted,  but,  with 
the  former,  difficulties  may  arise  in  consequence  of  the  presence  of  rapidly 
liquefying  colonies,  and  it  may  thus  be  necessary  to  count  after  twenty- 
four  hours. 

Probably  no  one  medium  will  support  the  growth  of  all  the  organisms 
present  in  a  given  sample  of  water,  and  under  certain  circumstances  special 
media  must  therefore  be  used.  Thus  Hansen  found  that  in  testing 
waters  to  be  used  in  brewing  it  was  advisable  to  have  in  the  medium 
employed  some  sterile  wort  or  beer,  so  that  the  organisms  in  the  test 
experiments  should  be  provided  with  the  food  materials  which  would  be 
present  in  the  commercial  use  of  the  water.  Manifestly  this  principle 
applies  generally  in  the  bacteriological  examination  of  waters  to  be  used 
for  industrial  purj>oses. 

Detection  of  the  Presence  of  Special  Organisms. — (a)  The  B.  coli  Group. — 
In  ordinary  public  health  work,  it  may  be  taken  that  the  most  frequent  and 
important  inquiry  with  regard  to  a  water  is  directed  to  the  investigation 
of  the  presence  or  absence  of  the  b.  coli  and  its  congeners.  Here  the 
method  adopted  is  to  determine  the  smallest  quantity  of  a  water  which 
gives  evidence  of  containing  organisms  of  this  type.  In  applying  any 
method  with  this  object  in  view  it  is,  we  consider,  absolutely  necessary 
that  it  shall  be  carried  out  at  the  spot  at  which  samples  are  collected. 

The  usual  method  is  to  use  as  the  primary  culture  medium  one  of  the 
bile-salt  preparations,  of  which  the  best  is  MacConkey's  bile-salt  glucose 
bouillon  to  which  litmus  has  been  added — glucose  being  used  in  prefer- 
ence to  lactose  in  order  to  bring  out  b.  enteritidis  of  Gaertner  if  this  be 
present.  In  this  medium  the  members  of  the  b.  coli  group  cause  changes 
n  suiting  in  the  formation  of  acid  and  gas.  It  is  thus  convenient  to  put 
the  nu-dium  into  Durham's  fermentation  tubes.  In  practice  we  employ 


158  BACTERIA  IN  WATER 

2-ouuce  cylindrical  mediciue  bottles,  4£  in.  high  by  1£  in.  in  diameter. 
The  medium,  along  with  the  inverted  test-tube,  is  placed  in  these  ; 
rubber  stoppers  are  inserted  in  the  mouths,  and  they  are  sterilised.  It  is 
customary  to  test  for  the  presence  of  the  organisms  in  any  sample  by 
adding  to  a  series  of  such  tubes  the  following  quantities  of  the,  water 
— 50  c.c.,  20c.  c.,  10  c.c.,  5  c.c.,  1  c.c.,  and,  it  may  be,  in  specially  sus- 
picious waters,  '5  c.c.,  -1  c.c.,  and  even  -01  c.c.  The  result  is  estimated 
in  terms  of  the  smallest  amount  of  water  with  which  the  occurrence  of 
acid  and  gas  formation  is  observed.  By  starting  with  a.  concentrated 
MacConkey's  mixture,  it  is  arranged  that,  when  the  sample  is  added,  the 
resulting  fluid  shall  be  of  the  concentration  of  MacConkey's  medium  as 
ordinarily  prepared.  Thus,  in  the  bottle  to  which  the  50  c.c.  sample  is 
to  be  added,  there  are  placed  10  c.c.  of  a  sixfold  concentration  of 
MacConkey's  medium.  In  the  20  c.c.  tube,  there  are  present  20  c.c.  of  a 
medium  of  double  strength  ;  in  the  10  c.c.  tube,  10  c.c.  of  a  mixture 
of  double  strength  ;  and  in  the  5  c.c.  tube,  5  c.c.  of  a  mixture  of  double 
strength.  With  smaller  samples,  we  simply  use  the  ordinary  MacConkey's 
medium. 

For  the  taking  of  the  samples,  sterile  8-ounce  stoppered  bottles  are 
convenient,  and  for  each  sample  it  is  necessary  to  have  sterile  25  c.c., 
10  c.c.  (graduated  to  tenths),  and  1  c.c.  (graduated  to  hundredths) 
pipettes.  The  armamentarium  being  thus  simple,  there  is.  no  difficulty 
in  carrying  out  the  necessary  manipulations  at  the  spot  where  the  sample 
is  collected. 

The  tubes  are  incubated  for  forty-eight  hours,  and  it  is  well  to  read  the 
results  at  the  end  of  the  first  twenty-four  hours  also.  The  formation  of 
acid  and  gas  in  the  tube  is  usually  recognised  as  "  presumptive  evidence  " 
of  the  presence  of  members  of  the  b.  coli  group,  but  it  is  necessary  to 
further  investigate  the  bacteria  giving  rise  to  this  change  to  determine 
whether  they  are  "typical"  or  "atypical"  b.  coli.  With  this  end  in 
view,  each  bottle  in  which  acid  and  gas  is  present  is  well  shaken  up,  two 
or  three  loopfuls  are  placed  on  a  plate  of  MacConkey's  neutral-red  bile-salt 
lactose  agar.  These  loopfuls  are  spread  over  the  surface  by  means  of  a 
sterile  spreader,  made  by  taking  a  piece  of  glass  rod  and  turning  a  portion 
about  2  inches  long  at  right  angles  to  the  shaft.  The  plates  are  incubated 
for  twenty-four  hours.  As  typical  b.  coli  produces  acid  in  lactose,  any 
colonies  of  such  an  organism  are  of  a  rosy  red  colour.  These  are  then 
picked  off,  sloped  agar  tubes  are  inoculated  and  used  for  the  further 
investigation  of  the  properties  of  the  bacterium  isolated. 

The  media  inoculated  should  be  gelatin  stab,  litmus  milk,  neutral-red 
lactose  bouillon,  glucose  broth,  peptone  water,  dulcite  peptone  water, 
adonite  peptone  water,  inuline  peptone  water,  saccharose  peptone  water, 
and  potato. 

It  is  well  in  dealing  with  the  neutral-red  lactose  agar  plates  to  inoculate 
a  lactose  peptone  water  tube  from  all  the  kinds  of  colonies  present, 
whether  these  are  red  or  not,  as  MacConkey  rightly  points  out  that  some- 
times an  organism  which  is  really  a  lactose  fermenter  does  not  produce 
a  red  colour  on  the  solid  medium.  There  is  another  point  to  be  noted 
here,  namely,  that  the  naked-eye  appearances  of  colonies  on  lactose  agar 
are  not  of  value  in  identifying  the  kind  of  organism  present. 

The  object  of  growing  suspicious  colonies  on  a  range  of  media  such  as 
that  given,  is  to  enable  typical  b.  coli  to  be  recognised  when  present.  At 
the  present  time  it  cannot  be  said  that  bacteriologists  are  in  agreement 
as  to  what  characters  determine  the  type  of  organism  most  frequently 
found  in  the  human  intestine — this,  of  course,  being  the  important  point 


BACTERIA  IN  WATER  159 

in  judging  of  the  contamination  of  a  water  supply.  The  subject  will  be 
more  fully  discussed  in  the  chapter  on  Typhoid  Fever.  Here  it  may  be 
said  that  for  work  on  water  two  attitudes  are  taken  up  in  this  country. 
I  ii>t.  that  of  Houston,  who  recognises  as  typical  qualities  the  following  : 
fluorescence  in  neutral  red  broth,  production  of  acid  and  gas  in  lactose 
peptone  water,  production  of  indol,  production  of  acid  and  clot  in  litmus 
milk  (so-called  "  flaginac "  reaction).  Secondly,  that  of  the  English 
Committee  of  1904,  which,  on  the  one  hand,  laid  stress  on  the  additional 
factor  of  non-liquefaction  of  gelatin,  and  on  the  other,  attached  less 
importance  to  the  production  of  indol  and  the  occurrence  of  fluorescence 
(see  p.  355). 

With  regard  to  saccharose  fermentation,  different  strains  of  coli  of 
undoubted  intestinal  origin  behave  differently  towards  saccharose,  but 
when  saccharose  is  fermented  the  occurrence  is  significant,  as  indicating 
a  great  probability  that  the  organism  is  intestinal  in  origin. 

(6)  B.  enteritidis  sporogcncs  and  streptococci. —  As  in  the  case  of 
sewage,  the  presence  of  these  in  a  water  may  be  sought  for.  The  methods 
are  those  which  have  already  been  given  (p.  154). 

Much  work  has  been  devoted  to  the  question  of  these  faical  streptococci 
presenting  specific  characters  by  which  they  could  be  differentiated 
from  other  streptococci.  Houston  has  found  that  the  prevailing  type 
of  organism  here  is  one  which  produces  acid  and  clot  in  milk,  reduces 
neutral-red,  and  ferments  saccharose,  lactose,  and  salicin.  It  corresponds 
to  the  streptococcus  fcecalis  of  Andrewes  and  Horder.  The  important 
point  in  this  connection  is  to  recognise  that  streptococci  of  such  a  type 
exist  in  great  numbers  in  human  faeces,  and  that  when  in  any  circum- 
stances faecal  contamination  is  suspected,  the  isolation  of  streptococci 
.strengthens  the  suspicion. 

With  regard  to  the  objects  with  which  the  bacteriological 
examination  of  water  may  be  undertaken,  though  these  may 
be  of  a  purely  scientific  character,  they  usually  aim  at  contribut- 
ing to  the  settlement  of  questions  relating  to  the  potability  of 
waters,  to  their  use  in  commerce,  and  to  the  efficiency  of 
processes  undertaken  for  the  purification  of  waters  which  have 
undergone  pollution.  The  last  of  these  objects  is  often  closely 
associated  with  the  first  two,  as  the  question  so  often  arises 
whether  a  purification  process  is  so  efficient  as  to  make  the 
water  again  fit  for  use. 

Water  derived  from  any  natural  source  contains  bacteria, 
though,  as  in  the  case  of  some  artesian  wells  and  some  springs,- 
the  numbers  may  be  very  small,  e.g.  4  to  100  per  c.c.  In  rain, 
snow,  and  ice  there  are  often  great  numbers,  those  in  the  first 
two  being  derived  from  the  air.  Great  attention  has  been  paid 
to  the  bacterial  content  of  wells  and  rivers.  With  regard  to 
the  former,  precautions  are  necessary  in  arriving  at  a  judgment. 
If  the  water  in  a  well  has  been  standing  for  some  time, 
multiplication  of  bacteria  may  give  a  high  value.  To  meet  this 
•  litlieulty  the  well  ought,  if  practicable,  to  be  pumped  dry  and 


160  BACTERIA  IN  WATER 

then  allowed  to  fill,  in  order  to  get  at  what  is  really  the  im- 
portant point,  namely,  the  bacterial  content  of  the  water 
entering  the  well.  Again,  if  the  sediment  of  the  well  has 
been  stirred  up,  a  high  value  is  obtained.  Ordinary  wells  of 
medium  depth  contain  from  100  to  2000  per  c.c.  With  regard 
to  rivers  very  varied  results  are  obtained.  Moorland  streams 
are  usually  fairly  pure.  In  an  ordinary  river  the  numbers 
present  vary  at  different  seasons  of  the  year,  whilst  the  pre- 
vailing temperature,  the  presence  or  absence  of  decaying 
vegetation,  or  of  washings  from  land,  and  dilution  with  large 
quantities  of  pure  spring  water,  are  other  important  features. 
Thus  the  Franklands  found  the  rivers  Thames  and  Lea  purest 
in  summer,  and  this  they  attributed  to  the  fact  that  in  this 
season  there  is  most  spring  water  entering,  and  very  little  water 
as  washings  off  land.  In  the  case  of  other  rivers  the  bacteria 
have  been  found  to  be  fewest  in  winter.  A  great  many  circum- 
stances must  therefore  be  taken  into  account  in  dealing  with 
mere  enumerations  of  water  bacteria,  and  such  enumerations 
are  only  useful  when  they  are  taken  simultaneously  over  a 
stretch  of  river,  with  special  reference  to  the  sources  of  the 
water  entering  the  river.  Thus  it  is  usually  found  that  im- 
mediately below  a  sewage  effluent  the  bacterial  content  rises, 
though  in  a  comparatively  short  distance  the  numbers  may 
markedly  decrease,  and  it  may  be  that  the  river  as  far  as 
numbers  are  concerned  may  appear  to  return  to  its  previous 
bacterial  content.  The  numbers  of  bacteria  present  in  rivers 
vary  so  greatly  that  there  is  little  use  in  quoting  figures,  most 
information  being  obtainable  by  comparative  enumerations  before 
and  after  a  given  event  has  occurred  to  a  particular  water. 
Such  a  method  is  thus  of  great  use  in  estimating  the  efficacy 
of  the  filter-beds  of  a  town  water  supply.  These  usually 
remove  from  95  to  98  per  cent,  of  the  bacteria  present,  and 
a  town  supply  as  it  issues  from  the  filter-beds  should  not 
contain  more  than  100  bacteria  per  c.c.  Again,  it  is  found 
that  the  storage  of  water  effects  a  very  marked  bacterial  purifica- 
tion. Thus  Houston  has  shown  in  one  series  of  observations 
that  while  93  per  cent,  of  samples  of  raw  river  Lea  water 
contained  b  coli.  in  1  c.c.  or  less,  in  the  stored  water  62  per 
cent,  of  the  samples  showed  no  b.  coli  to  be  present  in  100  c.c. 
The  highest  counts  of  bacteria  per  c.c.  are  observed  with  sewage  ; 
for  example,  in  the  London  sewage  the  numbers  range  from 
six  to  twelve  millions. 

Much   more   important   than   the    mere  enumeration  of  the 
bacteria  present  in  a  water  is  the  question  whether  these  include 


BACTERIA  IN  WATER  161 

forms  pathogenic  to  man.  The  chief  interest  here,  so  far  as 
Europe  is  concerned,  lies  in  the  fact  that  typhoid  fever  is  so 
frequently  water-borne,  but  cholera  and  certain  other  intestinal 
diseases  have  a  similar  source.  The  search  in  waters  for  the 
organisms  concerned  in  these  diseases  is  a  matter  of  the  greatest 
difficulty,  for  each  belongs  to  a  group  of  organisms  morpho- 
logically similar,  very  widespread  in  nature,  and  many  of  which 
have  little  or  no  pathogenic  action.  The  biological  characters 
of  these  organisms  will  be  given  in  the  chapters  devoted  to 
the  diseases  in  question,  but  here  it  may  be  said  that  from 
the  public  health  standpoint  the  making  of  their  being  found 
a  criterion  for  the  condemning  of  a  water  is  impracticable.  There 
is  no  doubt  that  the  typhoid  and  cholera  bacteria  can  exist 
for  some  time  in  water — at  least  this  has  been  found  to  be  the 
case  when  sterile  water  has  been  inoculated  with  these  bacteria. 
But  to  what  extent  the  same  is  true  when  they  are  placed  in 
natural  conditions,  which  involve  their  living  in  the  presence 
of  other  organisms,  is  unknown,  for  by  no  known  method  can 
the  presence  of  either  be  with  certainty  demonstrated  in  the 
complex  mixtures  which  occur  in  nature.  With  regard  to 
the  typhoid  bacillus,  of  late  the  tendency  has  been  to  seek  for 
the  presence  of  indirect  bacteriological  evidence  which  might 
point  in  the  direction  of  the  possibility  of  the  presence  of  this 
organism.  The  whole  question  turns  on  the  possibility  of 
recognising  bacteriologically  the  contamination  of  water  with 
<«•  wage.  Klein  and  Houston  here  insist  on  the  fact  that  in 
crude  sewage  the  b.  coli  -or  the  members  of  the  coli  group  are 
practically  never  fewer  than  100,000  per  c.c.,  and  therefore  if 
in  a  water  this  organism  forms  a  considerable  proportion  of  the 
total  number  of  organisms  present,  then  there  is  great  reason 
for  suspecting  sewage  pollution.  In  these  circumstances,  all 
modern  work  tends  to  taking  the  presence  of  b.  coli  in  a  water 
as  the  best  indirect  evidence  of  the  possibility  of  disease 
organisms  of  intestinal  origin  being  likely  to  gain  access  to 
that  water.  It  must,  however,  be  at  once  clearly  recognised  that 
the  presence  of  members  of  the  coli  group  is  only  an  indication, 
and  so  far  as  the  potability  of  any  water  is  concerned,  there 
is  no  evidence  that  these  organisms,  however  undesirable,  are 
under  ordinary  circumstances  actually  harmful  to  man.  In  all 
inquiries  there  is  the  difficulty  that  at  present  no  means  exist 
of  differentiating  between  b.  coli  as  derived  from  the  human 
inti-stine  on  the  one  hand,  and  from  the  intestine  of  animals 
on  the  other.  It  is  thus  necessary  in  reporting  upon  a  water  to 
havr  had  an  opportunity  of  inspecting  the  locality.  We  have 
1 1 


162  BACTERIA  IN  WATER 

known  cases  where  a  moorland  water  had  a  very  high  content 
in  b.  coli,  without  there  being  the  remotest  possibility 
that  such  came  from  man.  With  this  proviso,  we  must  inquire 
as  to  what  criteria  are  to  be  adopted  in  determining  the 
significance  of  the  presence  of  different  members  of  the  group 
in  a  water,  and  here  reliance  is  chiefly  to  be  placed  on  the 
presence  of  the  typical  forms  of  b.  coli. 

If  a  sufficient  quantity  of  practically  any  water  be  taken, 
except,  perhaps,  that  coming  from  artesian  wells,  organisms 
of  the  coli  group  will  be  found  to  be  present.  Therefore,  the 
question  resolves  itself  into  setting  up  some  standards  of 
relative  purity  which  may  be  followed  in  dealing  with  waters 
coming  from  different  sources.  These  standards  are  at  present 
empirical,  and  different  bacteriologists  have  different  views  on 
the  subject.  There  is,  however,  a  general  agreement  that  deep 
well  water  and  the  filtered  water  supplied  to  urban  communities 
should  be  entirely  free  from  b.  coli  in  quantities  of  100  c.c.  or 
less.  The  great  difficulty  lies  in  dealing  with  river  water  and 
water  from  shallow  and  surface  wells.  Here  the  usual  view 
is  that  the  presence  of  b.  coli  in  10  c.c.  or  less  is  sufficient  to 
condemn  the  water.  It  may  be  said  that  under  ordinary  circum- 
stances an  inspection  of  the  surroundings  and  an  unfavourable 
chemical  analysis  are  sufficient  to  condemn  such  a  water,  for  even 
if  a  bacteriological  examination  showed  the  absence  of  b.  coli 
in  large  samples,  yet  the  water  ought  to  be  condemned ;  and 
further,  if  in  a  suspicious  locality  the  bacteriological  analysis 
yielded  a  bad  result,  the  water  ought  to  be  condemned  even  if 
from  the  chemical  analysis  it  could  be  passed.  The  difficult 
cases  are  those  where  the  inspection  of  the  locality  is  satisfactory, 
and  yet  b.  coli  is  present  in  large  numbers.  'Here  contamination 
is  often  of  animal  origin,  and  the  water  can  after  careful  inquiry 
be  passed. 

Great  care  is  often  necessary  in  interpreting  bacteriological 
analysis  in  consequence  of  the  delicacy  of  the  method.  Thus 
in  examining  raw  waters,  especially  those  derived  from 
moorland  catchment  areas  to  be  used  for  urban  supplies, 
bacteriological  examinations  are  relatively  of  little  value,  as 
storage  and  filtration  will  completely  alter  the  bacterial  content. 
Bacteriological  methods  are,  however,  of  the  greatest  value — 
much  more  than  mere  chemical  analysis — in  determining  the 
efficiency  of  filtration  processes. 

As  the  b.  coli  is  fairly  widespread  in  nature,  Klein  and 
Houston  hold  that  valuable  supporting  evidence  is  found  in 
the  presence  of  the  b.  enteritidis  sporogenes  and  of  strepto- 


BACTERIOLOGY  OF  SEWAGE  163 

cocci,  both  of  which  are  probably  constant  inhabitants  of  the 
human  intestine.  The  spores  of  the  former  usually  number 
100  per  c.c.  in  sewage,  and  the  presence  of  the  latter  can  always 
be  recognised  in  '001  grm.  of  human  faeces.  The  deductions 
to  be  drawn  from  the  presence  of  these  in  water  are  the  same 
as  those  to  be  drawn  from  their  presence  in  soil. 

It  may  be  said  that  in  water  artificially  polluted  with  sewage 
containing  intestinal  bacteria,  these  can  be  detected  by  bacterio- 
logical methods  in  mixtures  from  ten  to  a  hundred  times  more 
dilute  than  those  in  which  the  pollution  can  be  detected  by 
purely  chemical  methods. 

Bacteriology  of  Sewage. — It  is  sometimes  necessary  to 
examine  the  bacterial  content  of  sewage,  especially  in  connection 
with  the  efficiency  of  purification  works.  The  main  lines  of 
inquiry  are  here  the  same  as  for  water,  and  the  general  methods 
are  identical,  the  only  modification  necessary  being  that,  in 
consequence  of  the  high  bacterial  content,  much  smaller 
quantities  of  the  raw  material  must  be  worked  with.  With 
regard  to  the  numbers  of  bacteria  in  sewage,  these  may  run 
from  a  million  to  ten  millions  or  even  more  per  c.c.,  and  here 
of  course  the  question  of  the  presence  of  intestinal  organisms 
of  the  coli  group  is  of  great  importance.  The  numbers  of  these 
are  large,  and  members  of  the  group  may  be  detected  in  a 
•000001  c.c.  or  less.  The  numbers  present  are  frequently 
considerably  reduced  by  purification  methods,  but  it  is  to  be 
noted  that,  even  when  such  methods  are  most  successful,  b.  coli 
may  yet  be  present  in  considerable  quantities.  This  is  especially 
true  in  Britain,  where  sewage  is  much  more  concentrated  than 
it  apparently  is  in  America.  In  the  latter  country,  purification 
may  yield  effluents  in  which  b.  coli  can  be  detected  in  only 
'001  c.c.  By  no  purification  method  has  the  production  of  a 
potable  water  been  attained,  and  the  high  content  of  effluents 
in  b.  coli  makes  the  passage  of  typhoid  bacilli  through  a  purifica- 
tion system  possible  although  the  organism  has  perhaps  never 
been  certainly  demonstrated. 

The  part  which  bacteria  play  in  the  purification  of  sewage 
constitutes  a  question  of  great  interest,  to  which  much  attention 
has  been  directed.  The  methods  adopted  for  sewage  purification 
may  be  divided  into  two  groups.  In  the  first  of  these,  the 
sewage  coming  from  the  mains  is  run  on  to  a  bed  of  gravel, 
clinker,  or  coke,  on  which  it  is  allowed  to  stand  for  some  hours. 
The  effluent  is  then  run  out  through  the  bottom  of  the  bed, 
which  is  then  allowed  to  rest  for  some  hours  before  being 
recharged.  In  a  modification  of  this  method  the  sewage  is 


164  BACTERIA  IN  WATER 

allowed  to  percolate  slowly  through  a  bed  consisting  of  large 
porous  objects,  such  as  broken  bricks  or  large  pieces  of  coke,  and 
here  the  percolation  may  be  constant,  no  interval  of  rest  being 
given.  The  bacterial  processes  which  take  place  in  these  two 
methods  are,  however,  probably  closely  similar.  In  the  second, 
the  essential  feature  is  a  preliminary  treatment  of  the 
sewage  in  more  or  less  closed  tanks  ("septic  tanks"),  where 
the  conditions  are  supposed  to  be  largely  anaerobic.  This 
method  has  been  adopted  at  Exeter,  Sutton,  and  Yeovil  in  this 
country,  and  very  fully  worked  at  in  America  by  the  State 
Board  of  Health  of  Massachusetts.  In  the  explanation  given 
of  the  rationale  of  this  process,  sewage  is  looked  on  as  exist- 
ing in  three  stages.  (1)  First  of  all,  fresh  sewage — the  newly 
mixed  and  very  varied  material  as  it  enters  the  main  sewers. 

(2)  Secondly,  stale  seivage  —  the  ordinary  contents  of  the  main 
sewers.     Here  there  is  abundant  oxygen,  and  as  the  sewage  flows 
along  there  occurs  by  bacterial   action  a  certain  formation  of 
carbon  dioxide  and  ammonia,  which  combine  to  form  ammonium 
carbonate.    This  is  the  sewage  as  it  reaches  the  purification  works. 
Here  a  preliminary  mechanical  screening  may  be  adopted,  after 
which    it    is    run    into    an    airtight   tank  —  the    septic    tank. 

(3)  It  remains  there  for  from  twenty-four  to  thirty-six  hours,  and 
becomes  a  foul-smelling  fluid — the  septic  sewage.     The  chemical 
changes  which  take  place  in  the  septic  tank  are  of  a  most  complex 
nature.     The  sewage  entering  it  contains  little  free  oxygen,  and 
therefore  the  bacteria  in  the  tank  are  probably  largely  anaerobic, 
and  the  changes  which  they  originate  consist  of  the  formation 
of  comparatively  simple  compounds  of  hydrogen  with  carbon, 
sulphur,  and  phosphorus.     As  a  result,  there  is  a  great  reduction 
in  the  amount  of  organic  nitrogen,  of  albuminoid  ammonia,  and 
of  carbonaceous  matter.     The  last  is  important,  as  the  clogging 
of  ordinary  filter-beds  is  largely  due  to  the  accumulation  of  such 
material,  and  of  matters  generally  consisting  of  cellulose.     One 
further  important  effect  is  that  the  size  of  the  particles  of  the 
deposited  matter  is  decreased,  and  therefore  it  is  more  easily  broken 
up  in  the  next  stage  of  the  process.     This  consists  of  running  the 
effluent  from  the  septic  tank  on  to  filter-beds,  preferably  of  coke, 
where  a  further  purification  process  takes  place.     By  this  method 
there  is  first  an  anaerobic  treatment,  succeeded  by  an  aerobic ; 
in  the  latter  the  process  of  nitrification  occurs  by  means  of  the 
special  bacteria  concerned.     The  results  are  of  a  satisfactory 
nature,  there  being  often  a  marked  diminution  in  the  number  of 
coli  organisms  present. 

In  the  earlier  stages  of  any  sewage  purification,  there  is  little 


BACTERIOLOGY  OF  SEWAGE  165 

doubt  that  the  albuminous  material  present  is  being  split  up  by 
ordinary  putrefactive  bacteria.  In  the  mains  and  where  open 
systems  of  purification  are  at  work,  aerobic  forms  play  the  chief 
part,  while  in  the  closed  methods  anaerobic  organisms  are  those 
chiefly  concerned.  In  contact  and  percolating  systems  there  is 
evidence  that  at  first  the  purifying  action  of  bacteria  is  materially 
furthered  by  physical  processes.  Thus  Dunbar  has  shown  that 
when  such  a  substance  as  coke  is  used  in  a  sewage  filter-bed  a 
considerable  amount  of  the  albuminous  material  is  removed  in 
a  very  few  minutes  by  adsorption,  for,  albumin,  being  of  a 
colloidal  nature,  is  readily  deposited  under  such  circumstances 
in  the  pores  of  the  coke  in  the  form  of  films.  After  a  time  such 
a  filter-bed  becomes  clogged,  but  on  access  of  oxygen  being 
allowed,  it  regains  its  adsorptive  properties — probably  from  the 
oxidation  of  the  material  adsorbed. 

During  this  stage,  as  in  the  whole  purification  process,  four, 
and  it  may  be  five,  processes  are  at  work  : — First,  the  action  of 
ordinary  bacteria  splitting  up  the  higher  albuminous  molecules ; 
secondly,  the  action  of  nitrifying  bacteria  building  up  nitrites 
and  nitrates  from  ammoniacal  products ;  thirdly,  the  action  of 
denitrifying  bacteria  which  reduce  nitrates  to  lower  gaseous 
oxides  and  to  free  nitrogen  (the  presence  of  which  in  filter  beds 
can  be  demonstrated) ;  fourthly,  the  action  of  higher  forms  of 
vegetable  and  animal  life ;  fifthly,  it  is  possible  that  direct 
chemical  oxidation  of  the  earlier  products  of  bacterial  action 
may  occur,  and  in  any  case  the  access  of  an  abundant  oxygen 
supply  to  adsorbed  material  hastens  its  destruction.  It  is 
possible,  as  is  indicated  by  the  work  of  Lorrain  Smith  and  of 
Mair,  that  perhaps  too  little  weight  has  been  attached  to  the 
parts  played  by  the  two  last  processes  specified,  for  in  the  later 
.stages  of  the  purification  process  there  is  a  very  marked 
diminution  in  the  number  of  bacteria  present  in  the  filter. 
Much  further  work,  however,  is  necessary  before  the  part  to  be 
assigned  to  each  factor  in  operation  can  be  properly  estimated. 

Further,  the  details  of  the  essentially  bacterial  part  of  the 
process  are  obscure,  and  the  relative  parts  played,  even  in  an 
open  purification  process,  by  aerobes  on  the  one  hand,  and 
anaerobes  on  the  other,  is  little  understood.  When  sewage  is 
drained  off  to  rest  a  filter-bed,  great  quantities  of  oxygen  are 
sucked  in,  but  as  to  how  long  the  bed  thus  remains  aerated, 
authorities  differ — some  maintaining  that  oxidation  processes  per- 
sist even  after  the  bed  has  been  recharged,  while  others  state  that 
the  oxygen  in  the  resting  bed  is  consumed,  and  its  place 
by  carbon  dioxide  and  nitrogen.  Certainly,  at  certain 


166  ANTISEPTICS 

stages  of  the  purification  process,  large  amounts  of  free  nitrogen 
come  off  the  bed,  but  whether  at  such  periods  anaerobic  bacteria 
are  or  are  not  in  the  ascendant,  is  not  known.  It  is  probable 
that,  from  the  practical  standpoint,  the  later  stages  of  purification 
should  take  place  with  free  oxidation,  as  when  anaerobic  bacteria 
are  active  at  this  point  a  very  offensive  effluent  is  produced. 

Often  the  effluent  from  a  sewage  purification  system  contains 
as  many  bacteria  as  the  sewage  entering,  but  there  is  often  a 
marked  diminution.  It  is  said  by  some  that  pathogenic 
bacteria  do  not  live  in  sewage.  The  typhoid  bacillus  has  been 
found  to  die  out  when  placed  in  sewage,  but  it  certainly  can 
live  in  this  fluid  for  a  much  longer  period  than  that  embraced 
by  any  purification  method.  Thus  the  constant  presence  of 
b.  coli,  b.  enteritidis,  and  streptococci  which  has  been  observed 
in  sewage  effluents  must  here  still  be  looked  on  as  indicating  a 
possible  infection  with  the  typhoid  bacillus,  and  it  is  only  by 
great  dilution  and  prolonged  exposure  to  the  conditions  present 
in  running  water  that  such  an  effluent  can  become  suitable  for 
forming  a  part  of  a  potable  water. 

ANTISEPTICS. 

The  death  of  bacteria  is  judged  of  by  the  fact  that,  when 
they  are  placed  on  a  suitable  food  medium,  no  development 
takes  place.  Microscopically  it  would  be  observed  that  division 
no  longer  occurred,  and  that  in  the  case  of  motile  species  move- 
ment would  have  ceased,  but  such  an  observation  has  only 
scientific  interest.  From  the  importance  of  being  able  to  kill 
bacteria,  an  enormous  amount  of  work  has  been  done  in  the  way 
of  investigating  the  means  of  doing  so  by  chemical  means,  and 
the  bodies  having  such  a  capacity  are  called  antiseptics.  It  is 
now  known  that  the  activity  of  these  agents  is  limited  to  the 
killing  of  bacteria  outside  the  animal  body,  but  still  even  this  is 
of  high  importance. 

Methods. — These  vary  very  much.  In  early  inquiries  a  great  point 
was  made  of  the  prevention  of  putrefaction,  and  work  was  done  in  the 
way  of  finding  how  much  of  an  agent  must  be  added  to  a  given  solution 
such  as  beef  extract,  urine,  etc.,  in  order  that  the  bacteria  accidentally 
present  might  not  develop  ;  but  as  bacteria  vary  in  their  powers  of  re- 
sistance, the  method  was  unsatisfactory,  and  now  an  antiseptic  is  usually 
judged  of  by  its  effects  on  pure  cultures  of  definite  pathogenic  microbes, 
and  in  the  case  of  a  sporing  bacterium,  the  effect  on  both  the  vegetative 
and  spore  forms  is  investigated.  The  organisms  most  used  are  the 
staphylococcus  pyogenes,  streptococcus  pyogenes,  and  the  organisms  of 
typhoid,  cholera,  diphtheria,  and  anthrax— the  latter  being  most  used 


ANTISEPTICS  167 

for  testing  the  action  on  spores.  The  best  method  to  employ  is  to  take 
sloped  agar  cultures  of  the  test  organism,  scrape  off  the  growth,  and  mix 
it  up  with  a  small  amount  of  distilled  water,  and  filter  this  emulsion 
through  a  plug  of  sterile  glass  wool  held  in  a  .small  sterile  glass  funnel, 
add  a  measured  quantity  of  this  fluid  to  a  given  quantity  of  a  solution 
of  the  antiseptic  in  distilled  water,  then  after  the  lapse  of  the  period  of 
observation  to  remove  one  or  two  loopfuls  of  the  mixture  and  place  them 
in  a  great  excess  of  culture  medium.  Here  it  is  preferable  to  use  fluid 
agar,  which  is  then  plated  and  incubated  ;  such  a  procedure  is  preferable 
to  the  use  of  bouillon  tubes,  as  any  colonies  developing  can  easily  be 
recognised  as  belonging  to  the  species  of  bacterium  used.  In  dealing 
with  strong  solutions  of  chemical  agents  it  is  necessary  to  be  sure  that 
the  culture  fluid  is  in  great  excess,  so  that  the  small  amount  of  the 
antiseptic  which  is  transferred  with  the  bacteria  may  be  diluted  far 
beyond  the  strength  at  which  it  still  can  have  any  noxious  influence. 
Sometimes  it  is  possible  at  the  end  of  the  period  of  observation  to 
change  the  antiseptic  into  inert  bodies  by  the  addition  of  some  other 
substance,  and  then  test  the  condition  of  the  bacteria,  and  if  the  inert 
substances  are  fluid  there  is  no  objection  to  this  proceeding  ;  but  if  in 
the  process  a  precipitate  results,  then  it  is  better  not  to  have  recourse 
to  such  a  method,  as  sometimes  the  bacteria  are  carried  down  with  the 
precipitate  and  may  escape  the  culture  test.  The  advisability  of,  when 
possible,  thus  chemically  changing  the  antiseptic  was  first  brought  to 
notice  by  the  criticism  of  Koch's  statements  as  to  the  efficacy  of 
mercuric  chloride  in  killing  the  spores  of  the  b.  anthracis.  The  method 
he  employed  in  his  experiments  was  to  soak  silk  threads  in  an  emulsion 
of  anthrax  spores  and  dry  them.  These  were  then  subjected  to  the 
action  of  the  antiseptic,  well  washed  in  water,  and  laid  on  the  surface  of 
agar.  It  was  found,  however,  that,  with  threads  exposed  to  a  far  higher 
concentration  of  the  corrosive  sublimate  than  Koch  had  stated  was 
sufficient  to  prevent  growth,  if  the  salt  were  broken  up  by  the  action  of 
ammonium  sulphide  and  this  washed  off,  growth  of  anthrax  still  occurred 
when  the  threads  were  laid  on  agar.  The.  explanation  given  was  that 
the  antiseptic  had  formed  an  albuminate  with  the  case  of  each  spore,  and 
that  this  prevented  the  antiseptic  from  acting  upon  the  contained 
protoplasm.  Such  an  occurrence  only  takes  place  with  spores,  and  the 
method  given  above,  in  which  the  small  amount  of  antiseptic  adhering 
to  the  bacteria  is  swamped  in  an  excess  of  culture  fluid,  can  safely  be 
followed,  especially  when  a  series  of  antiseptics  is  being  compared. 
Kro'nig  and  Paul  introduced  what  is  known  as  the  Garnet  method  for 
testing  antiseptics.  In  this,  small  garnets  of  equal  size  are  carefully 
cleaned,  dipped  in  an  emulsion  of  anthrax  spores,  and  allowed  to  dry. 
They  are  then  placed  in  mercuric  chloride,  and  from  time  to  time  some 
are  removed,  gently  washed,  and  treated  with  ammonium  sulphide  to 
decompose  the  chloride.  They  are  then  well  shaken  in  a  measured 
quantity  of  water.  This  is  plated,  and  the  number  of  anthrax  colonies 
developing  is  counted. 

Ponder  and  Woodhead  have  introduced  an  ingenious  apparatus  by 
which  the  effects  of  different  concentrations  of  an  antiseptic  on  the 
vitality  of  such  an  organism  as  the  b.  coli  can  be  automatically 
recorded. 

Much  attention  has  been  paid  to  the  standardisation  of  antiseptics, 
and  a  watery  solution  of  carbolic  acid  is  now  generally  taken  as  the 
standard  with  which  other  antiseptics  are  compared.  Rideal  and 
Walker  point  out  that  110  parts  by  weight  of  B.P.  carbolic  acid  equal 


168  ANTISEPTICS 

100  parts  by  weight  of  phenol,  and  they  recommend  the  following  method 
of  standardising:  To  5  c.c.  of  a  particular  dilution  of  the  disinfectant 
add  5  drops  of  a  24-hour-old  bouillon  culture  of  the  organism  (usually 
b.  typhosus),  which  has  been  incubated  at  37°  C.  Shake  the  mixture 
and  make  subcultures  every  2£  minutes  to  15  minutes.  Perform  a 
parallel  series  of  experiments  with  carbolic  acid,  and  express  the 
comparative  result  in  multiples  of  the  carbolic  acid  doing  the  same 
work. 

The  Action  of  Antiseptics. — In  inquiries  into  the  actions 
of  antiseptics  attention  to  a  great  variety  of  factors  is  necessary, 
especially  when  the  object  is  not  to  compare  different  antiseptics 
with  one  another,  but  when  the  absolute  value  of  any  body  is 
being  investigated.  Thus  the  medium  in  which  the  bacteria  to 
be  killed  are  situated  is  important ;  the  more  albuminous  it 
is,  the  greater  degree  of  concentration  is  required.  Again, 
the  higher  the  temperature  at  which  the  action  is  to  take 
place,  the  more  dilute  may  the  antiseptic  be,  or  the  shorter  the 
exposure  necessary  for  a  given  effect  to  take  place.  The  most 
important  factor,  however,  to  be  considered  is  the  chemical 
nature  of  the  substances  employed.  Chick  has  shown  that  the 
action  of  a  disinfectant  upon  a  bacterium  presents  close 
analogies  with  the  interaction  of  simple  chemical  substances, 
such  as  an  acid  and  an  alkali.  In  the  case  of  anthrax  spores, 
during  the  first  few  minutes  a  great  fatality  occurs,  after  which 
the  action  of  the  antiseptic  gradually  tails  off.  With  certain 
other  organisms,  however,  such  as  the  paratyphoid  bacillus, 
the  presence  in  a  culture — especially  in  a  young  culture — of 
highly  resistant  forms  renders  the  initial  action  of  an  antiseptic 
less  marked.  The  action  of  an  antiseptic,  like  the  action  of  an 
acid  and  an  alkali,  is  very  much  increased  by  raising  the 
temperature ;  from  which  follows  the  practical  conclusion  that, 
in  any  disinfection,  the  use  of  warm  solutions  is  advisable. 
Chick  and  C.  J.  Martin  have  further  investigated  the  fact  that 
the  presence  of  albuminous  material  in  a  mixture  of  disinfectant 
and  bacteria  decreases  the  action  of  the  disinfectant,  and 
consider  that  the  latter  is  adsorbed  by  the  albumin.  They  have 
shown  grounds  for  believing  that  a  disinfectant  in  an  emulsion- 
ised  form  is  more  efficient  than  a  similar  disinfectant  in  actual 
solution,  because  of  a  similar  phenomenon  occurring ;  for, 
just  as  a  disinfectant  may  be  put  out  of  action  by  being 
adsorbed  by  organic  particles,  so  when  these  organic  particles 
happen  to  be  bacteria,  the  adsorption  process  causes  a  greater 
concentration  of  the  antiseptic  round  the  bacterial  protoplasm, 
and  thus  hastens  its  death. 

Though  nearly  every  substance  which  is  not  a  food  to  the 


THE  ACTION  OF  ANTISEPTICS  169 

animal  or  vegetable  body  is  more  or  less  harmful  to  bacterial 
life,  yet  certain  bodies  have  a  more  marked  action  than  others. 
Thus  it  may  be  said  that  the  most  important  antiseptics  are  the 
salts  of  the  heavy  metals,  certain  acids,  especially  mineral  acids, 
certain  oxidising  and  reducing  agents,  a  great  variety  of  sub- 
stances belonging  to  the  aromatic  series,  and  volatile  oils  generally. 
In  comparing  different  bodies  belonging  to  any  one  of  these 
groups  the  chemical  composition  or  constitution  is  very  important, 
and  if  such  comparisons  are  to  be  made,  the  solutions  compared 
must  be  equimolecular  ;  in  other  words,  the  action  of  a  molecule 
of  one  body  must  be  compared  with  the  action  of  a  molecule  of 
another  body.  This  can  be  done  by  dissolving  the  molecular 
weight  in  grammes  in,  say,  a  litre  of  water  (see  p.  34).  When 
this  is  done,  important  facts  emerge.  Thus,  generally  speaking, 
the  compounds  of  a  metal  of  high  atomic  weight  are  more 
powerful  antiseptics  than  those  of  one  belonging  to  the  same 
series,  but  of  a  lower  atomic  weight.  Among  organic  bodies, 
;iur  tin,  substances  with  high  molecular  weight  are  more  powerful 
than  those  of  low  molecular  weight — thus  butyric  alcohol  is  more 
powerful  than  ethylic  alcohol — and  important  differences  among 
the  aromatic  bodies  are  associated  with  their  chemical  constitu- 
tion. Thus  among  the  cresols  the  ortho-  and  para-bodies  re- 
semble each  other  in  general  chemical  properties,  and  stand  apart 
from  metacresol ;  they  also  are  similar  in  antiseptic  action,  and 
are  much  stronger  than  the  meta-body.  The  same  may  be 
observed  in  other  groups  of  ortho-,  meta-,  and  para-bodies. 
Again,  such  a  proj>erty  as  acidity  is  important  in  the  action  of  a 
substance,  and,  generally  speaking,  the  greater  the  avidity  of  an 
acid  to  combine  with  an  alkali,  the  more  powerful  an  antiseptic 
it  is.  With  regard  to  oxidising  agents  and  reducing  agents, 
probably  the  possession  of  such  properties  has  been  overrated  as 
increasing  bactericidal  potency.  Thus  in  the  case  of  such  re- 
ducers as  sulphurous  acid  and  formic  acid,  the  effect  is  apparently 
chiefly  due  to  the  fact  that  these  substances  are  acids.  Formic 
acid  is  much  more  efficient  than  formate  of  sodium.  In  the  case 
of  permanganate  of  potassium,  which  is  usually  taken  as  the 
type  of  oxidising  agents  in  this  connection,  it  can  be  shown  that 
the  greater  amount  of  the  oxidation  which  takes  place  when  this 
agent  is  brought  into  contact  with  bacteria  occurs  after  the 
organisms  are  killed.  Such  an  observation  is,  however,  not 
conclusive  as  to  the  non-efficiency  of  the  oxidation  process,  t  < » 
the  death  of  the  bacteria  might  be  due  to  the  oxidation  of  a 
\<TY  small  part  of  the  bacterial  protoplasm.  Apart  from  the 
chemical  nature  of  antiseptic  agents,  the  physical  factors  con- 


170  ANTISEPTICS 

cerned  in  their  solution,  especially  when  they  are  electrolytes, 
probably  play  a  part  in  their  action.  The  part  played  by  such 
factors  is  exemplified  in  the  important  fact  that  a  strong  solution 
acting  for  a  short  time  will  have  the  same  effect  as  a  weaker 
solution  acting  for  a  longer  time.  From  what  has  been  said  it 
will  be  realised  that  the  real  causes  of  a  material  being  an 
antiseptic  are  very  obscure,  and  at  present  we  can  only  have  a 
remote  idea  of  the  factors  at  work. 

The  Effects  of  certain  Antiseptics. — Here  we  can  only 
briefly  indicate  certain  results  obtained  with  the  more  common 
members  of  the  group. 

Chlorine. — All  the  halogens  have  been  found  to  be  powerful 
antiseptics,  but  from  the  cheapness  with  which  it  can  be  produced 
chlorine  has  been  most  used ;  not  only  is  it  the  chief  active 
agent  in  the  somewhat  complex  action  of  bleaching  powder,  but 
it  is  also  the  chief  constituent  of  several  proprietary  substances, 
of  which  "  Electrozone  "  is  a  good  example.  This  last  substance 
is  made  from  electrolysing  sea- water,  when  magnesia,  and  chlorine 
being  liberated,  magnesium  hypochlorite  and  magnesium  chloride 
are  formed.  In  the  action  of  this  substance  free  hypochlorous 
acid  is  formed,  and  the  effect  produced  is  thus  similar  to  that 
of  bleaching  powder.  Nissen,  investigating  the  action  of  the 
latter,  found  that  1J  per  cent,  killed  typhoid  bacilli  in  faeces  ; 
and  Rideal  found  that  1  part  to  400-500  disinfected  sewage  in 
fourteen  minutes,  and  Delepine's  results  show  that  1  part  to  50 
(equal  to  '66  per  cent,  of  chlorine)  rapidly  kills  the  tubercle 
bacillus,  and  1  part  to  10  (equal  to  3*3  per  cent.)  killed  anthrax 
spores.  Klein  found  that  '05  per  cent,  of  chlorine  killed  most 
bacterial  spores  in  five  minutes. 

Iodine  Terchloride. — This  is  a  very  unstable  compound  of 
iodine  and  chlorine,  and,  seeing  that  the  substance  only  remains 
as  IC13  in  an  atmosphere  of  chlorine  gas,  it  is  open  to  doubt 
whether  the  antiseptic  effects  attributed  to  it  are  not  due  to  a 
very  complicated  action  of  free  hydrochloric  acid,  hydriodic  acid, 
of  oxyacids  of  chlorine  and  iodine  produced  by  its  decomposition, 
and  also,  in  certain  cases,  of  organic  iodine  compounds  formed 
from  its  contact  with  albuminous  material.  It  is  stated  that  the 
action  is  very  potent :  a  1  per  cent,  solution  is  said  instantly  to 
kill  even  anthrax  spores,  but  if  the  spores  be  in  bouillon,  death 
occurs  after  from  ten  to  twelve  minutes.  In  serum  the  necessary 
exposure  is  from  thirty  to  forty  minutes.  A  solution  of  1-1000 
will  kill  the  typhoid,  cholera,  and  diphtheria  organisms  in  five 
minutes. 

Nascent  Oxygen. — This  is  chiefly  available  in  two  ways — firstly, 


THE  EFFECTS  OF  CERTAIN  ANTISEPTICS      171 

when  in  the  breaking  up  of  ozone  the  free  third  atom  of  the 
ozone  molecule  is  seeking  to  unite  with  another  similar  atom ; 
secondly,  when  peroxide  of  hydrogen  is  broken  up  into  water 
and  an  oxygen  atom  is  thereby  liberated.  In  commerce  the 
activity  of  "Sanitas"  compounds  is  due  to  the  formation  of 
ozone  by  the  slow  oxidation  of  the  resin,  camphor,  and  thymol 
they  contain. 

Perchloride  of  Mercury. — Of  all  the  salts  of  the  heavy  metals 
this  has  been  most  widely  employed,  and  must  be  regarded  as 
one  of  the  most  powerful  and  useful  of  known  antiseptics.  In 
testing  its  action  on  anthrax  spores  there  is  no  doubt  that  in  the 
earlier  results  its  potency  was  overrated  from  a  neglect  of  the 
fact  already  alluded  to,  that  in  the  spore-case  an  albuminate  of 
mercury  was  formed  which  prevented  the  contained  protoplasm 
from  developing,  while  not  depriving  it  of  life.  It  has  been 
found,  however,  that  this  salt  in  a  strength  of  1-100  will  kill  the 
spores  in  twenty  minutes,  although  an  hour's  exposure  to  1-1000 
has  no  effect.  The  best  results  are  obtained  by  the  addition  to 
the  corrosive  sublimate  solution  of  '5  per  cent,  of  sulphuric  acid 
or  hydrochloric  acid ;  the  spores  will  then  be  killed  by  a  seventy- 
minute  exposure  to  a  1-200  solution.  When,  however,  organisms 
in  the  vegetative  condition  are  being  dealt  with,  much  weaker 
solutions  are  sufficient;  thus  anthrax  bacilli  in  blood  will  be 
killed  in  a  few  minutes  by  1-2000,  in  bouillon  by  1-40,000,  and 
in  water  by  1-500,000.  Plague  bacilli  are  killed  by  one  to  two 
minutes'  exposure  to  1-3000.  Generally  speaking,  it  may  be  said 
that  a  1-2000  solution  must  be  used  for  the  practically  instan- 
taneous killing  of  vegetative  organisms. 

Perchloride  of  mercury  is  one  of  the  substances  which  have 
been  used  for  disinfecting  rooms  by  distributing  it  from  a  spray 
producer,  of  which  the  Equifex  may  be  taken  as  a  type.  With 
such  a  machine  it  is  calculated  that  1  oz.  of  perchloride  of 
mercury  used  in  a  solution  of  1-1000  will  probably  disinfect  3000 
square  feet  of  surface.  Such  a  procedure  has  been  extensively 
used  in  the  disinfection  of  plague  houses,  but  the  use  of  a  stronger 
solution  (1-500  acidulated)  is  probably  preferable. 

Formalin  as  a  commercial  article  is  a  40  per  cent,  solution  of 
formaldehyde  in  water.  This  is  a  substance  which  of  late  years 
has  come  much  into  vogue,  and  it  is  undoubtedly  a  valuable 
antiseptic.  A  disadvantage,  however,  to  its  use  is  that,  when 
diluted  and  exposed  to  air,  amongst  other  changes  which  it 
undergoes  it  may  be  transformed,  under  little  understood 
conditions,  into  trioxymethylene  and  paraformaldehyde,  these 
being  polymers  of  formaldehyde.  The  bactericidal  values  of  these 


172  ANTISEPTICS 

mixtures  are  thus  indefinite.  Formalin  may  be  used  either  by 
applying  it  in  its  liquid  form  or  as  a  spray,  or  the  gas  which 
evaporates  at  ordinary  temperatures  from  the  solution  may  be 
utilised.  To  disinfect  such  an  organic  mixture  as  pus  containing 
pyogenic  organisms,  a  10  per  cent,  solution  acting  for  half  an 
hour  is  necessary.  In  the  case  of  pure  cultures,  a  5  per  cent, 
solution  will  kill  the  cholera  organism  in  three  minutes,  anthrax 
bacilli  in  a  quarter  of  an  hour,-  and  the  spores  in  five  hours. 
When  such  organisms  as  pyogenic  cocci,  cholera  spirillum,  and 
anthrax  bacillus  infect  clothing,  an  exposure  to  the  full  strength 
of  formalin  for  two  hours  is  necessary,  and  in  the  case  of  anthrax 
spores,  for  twenty-four  hours.  Silk  threads  impregnated  with 
the  plague  bacillus  were  found  to  be  sterile  after  two  minutes' 
exposure  to  formalin. 

The  action  of  formalin  vapour  has  been  much  studied,  as  its 
use  constitutes  a  cheap  method  of  treating  infected  rooms,  in 
which  case  some  spray-producing  machine  is  employed.  It  is 
stated  that  a  mixture  of  8  c.c.  of  formalin  with  48  c.c.  of  water 
is  sufficient  when  vaporised  to  disinfect  one  cubic  metre,  so  far 
as  non-sporing  organisms  are  concerned.  It  is  also  stated  that 

I  part  formalin  in  10,000  of  air  will  kill  the  cholera  vibrio  in 
one  hour,  diphtheria  bacillus  in  three  hours,  the  staphylococcus 
pyogenes  in  six  hours,  and  anthrax  spores  in  thirteen  hours.     In 
the  .case  of  organisms  which  have   become  dry  it  is  probable, 
however,  that  much  longer  exposures  are  necessary,  but  on  this 
point  we  have  not  definite  information. 

Formalin  gas  has  only  a  limited  application ;  it  has  little 
effect  on  dry  organisms,  and  in  the  case  of  wet  organisms,  in 
order  to  be  effective,  probably  must  become  dissolved  so  as  to 
give  the  moisture  a  proportion  analogous  to  the  strengths  stated 
above  with  regard  to  the  vapour. 

Sulphurous  Acid. — This  substance  has  long  been  in  use, 
largely  from  the  cheapness  with  which  it  can  be  produced  by 
burning  sulphur  in  the  air.  An  atmosphere  containing  -98  per 
cent,  will  kill  the  pyogenic  cocci  in  two  minutes  if  they  are  wet, 
and  in  twenty  minutes  if  they  are  dry ;  and  anthrax  bacilli  are 
killed  by  thirty  minutes'  exposure,  but  to  kill  anthrax  spores  an 
exposure  of  from  one  to  two  hours  to  an  atmosphere  containing 

I 1  per  cent,  is  necessary.     For  a  small  room  the  burning  of  about 
a  pound  and  a  half  (most  easily  accomplished  by  moistening  the 
sulphur  with  methylated  spirit)  is  usually  considered  sufficient. 
It  has  been  found  that  if  bacteria  are  protected,  e.g.  when  they 
are   in   the   middle  of    small   bundles    of   clothes,  no    effect   is 
produced  even  by  an  atmosphere  containing  a  large  proportion 


THE  EFFECTS  OF  CERTAIN  ANTISEPTICS      173 

of  the  sulphurous  acid  gas.  The  practical  applications  of  this 
agent  are  therefore  limited. 

Potassium  Permanganate. — The  action  of  this  agent  very 
much  depends  on  whether  it  can  obtain  free  access  to  the 
bacteria  to  be  killed  or  whether  these  are  present  in  a  solution 
containing  much  organic  matter.  In  the  latter  case  the  oxidation 
of  the  organic  material  throws  so  much  of  the  salt  out  of  action 
that  there  may  be  little  left  to  attack  the  organisms.  Koch 
found  that  to  kill  anthrax  spores  a  5  per  cent,  solution  required 
to  act  for  about  a  day ;  for  most  organisms  a  similar  solution 
acting  for  shorter  periods  has  been  found  sufficient,  and  in  the 
cases  of  the  pyogenic  cocci  a  1  per  cent,  solution  will  kill  in  ten 
minutes.  There  is  little  doubt  that  such  weaker  solutions  are  of 
value  in  disinfecting  the  throat  on  account  of  their  non-irritating 
properties,  and  good  results  in  this  connection  have  been  obtained 
in  cases  of  diphtheria.  A  solution  of  1  in  10,000  has  been  found 
to  kill  plague  bacilli  in  five  minutes. 

Carbolic  Acid. — Of  all  the  aromatic  series  this  is  the  most 
extensively  employed  antiseptic.  All  ordinary  bacteria  in  the 
vegetative  condition,  and  of  these  the  staphylococcus  pyogenes 
is  the  most  resistant,  are  killed  in  less  than  five  minutes  by  a 
2-3  per  cent,  solution  in  water,  so  that  the  5  per  cent,  solution 
usually  employed  in  surgery  leaves  a  margin  of  safety.  But  for 
the  killing  of  such  organisms  as  anthrax  spores  a  very  much 
longer  exposure  is  necessary ;  thus  Koch  found  it  necessary  to 
expose  these  spores  for  four  days  to  ensure  disinfection.  The 
risk  of  such  spores  being  present  in  ordinary  surgical  procedure 
may  be  overlooked,  but  there  might  be  risk  of  tetanus  spores 
not  being  killed,  as  these  will  withstand  fifteen  hours'  exposure 
to  a  5  per  cent,  solution. 

In  the  products  of  the  distillation  of  coal  there  occur,  besides 
carbolic  acid,  many  bodies  of  a  similar  chemical  constitution,  and 
many  mixtures  of  these  are  in  the  market — the  chief  being  cyllin, 
izal,  and  lysol,  all  of  which  are  agents  of  value.  Of  these  lysol 
is  perhaps  the  most  noticeable,  as  from  its  nature  it  acts  as  a 
soap,  and  thus  can  remove  fat  and  dirt  from  the  hands.  A  one- 
third  per  cent,  solution  is  said  to  destroy  the  typhoid  and  cholera 
organisms  in  twenty  minutes.  A  one  per  cent,  solution  is  sufficient 
for  ordinary  surgical  procedures. 

lodoform. — This  is  an  agent  regarding  the  efficacy  of  which 
there  has  been  much  dispute.  There  is  little  doubt  that  it  owes 
its  efficiency  to  its  capacity  for  being  broken  up  by  bacterial 
action  in  such  a  way  as  to  set  free  iodine,  which  acts  as  a 
powerful  disinfectant.  The  substance  is  therefore  of  value  in  the 


174  ANTISEPTICS 

treatment  of  foul  wounds,  such  as  those  of  the  mouth  and 
rectum,  where  reducing  bacteria  are  abundantly  present.  It  acts 
more  slightly  where  there  are  only  pyogenic  cocci,  and  it  seems 
to  have  a  specially  beneficial  effect  in  tubercular  affections.  In 
certain  cases  its  action  may  apparently  be  aided  by  the  presence 
of  the  products  of  tissue  degeneration. 

From  the  results  which  have  been  given  it  will  easily  be 
recognised  that  the  choice  of  an  antiseptic  and  the  precise 
manner  in  which  it  is  to  be  employed  depend  entirely  on  the 
environment  of  the  bacteria  which  are  to  be  killed.  In  many 
cases  it  will  be  quite  impossible,  without  original  inquiry,  to  say 
what  course  is  likely  to  be  attended  with  most  success. 


CHAPTER  VI. 

RELATIONS  OF  BACTERIA  TO  DISEASE— THE 
PRODUCTION  OF  TOXINS  BY  BACTERIA. 

Introductory. — It  has  already  been  stated  that  a  strict  division 
of  micro  organisms  into  saprophytes  and  true  parasites  cannot  be 
made.  No  doubt  there  are  organisms,  such  as  the  bacillus  of 
leprosy,  which  as  yet  have  not  been  cultivated  outside  the  animal 
body,  and  others,  such  as  the  gonococcus,  which  are  in  natural 
conditions  always  parasites  associated  with  disease.  But  these 
latter  can  lead  a  saprophytic  existence  in  specially  prepared 
conditions,  and  there  are  many  of  the  disease  -  producing 
organisms,  such  as  the  organisms  of  typhoid  and  cholera,  which 
can  nourish  readily  outside  the  body,  even  in  ordinary  con- 
ditions. The  conditions  of  growth  are,  however,  of  very  great 
importance  in  the  study  of  the  modes  of  infection  in  the  various 
diseases,  though  they  do  not  form  the  basis  of  a  scientific 
division. 

A  similar  statement  applies  to  the  terms  pathogenic  and 
saprophytic,  and  even  to  the  terms  pathogenic  and  non-patho- 
genic. By  the  term  pathogenic  is  meant  the  power  which  an 
organism  has  of  producing  morbid  changes  or  effects  in  the 
animal  body,  either  under  natural  conditions  or  in  conditions 
artificially  arranged,  as  in  direct  experiment.  Now  we  know  of 
no  organisms  which  will  in  all  circumstances  produce  disease  in 
all  animals,  and,  on  the  other  hand,  many  bacteria  described  as 
harmless  saprophytes  will  produce  pathological  changes  if  intro- 
duced in  sufficient  quantity.  When,  therefore,  we  speak  of  a 
pathogenic  organism,  the  term  is  merely  a  relative  one,  and 
indicates  that  in  certain  circumstances  the  organism  will  produce 
disease,  though  in  the  science  of  human  pathology  it  is  often 
used  for  convenience  as  implying  that  the  organism  produces 
disease  in  man  in  natural  conditions. 

Modifying  Conditions. — In  studying  the  pathogenic  effects  in 
any  instance,  both  the  micro-organisms  and  the  animal  affected 

175 


176        RELATIONS  OF  BACTERIA  TO  DISEASE 

must  be  considered,  and  not  only  the  species  of  each,  but  also 
its  exact  condition  at  the  time  of  infection.  In  other  words, 
the  resulting  disease  is  the  product  of  the  sum-total  of  the 
characters  of  the  infecting  agent,  on  the  one  hand,  and  of  the 
subject  of  infection,  on  the  other.  We  may,  therefore,  state 
some  of  the  chief  circumstances  which  modify  each  of  these 
two  factors  involved,  and,  consequently,  the  diseased  condition 
produced. 

1.  The  Infecting  Agent. — In  the  case  of  a  particular  species 
of  bacterium  its  effect  will  depend  chiefly  upon  (a)  its  virulence, 
and  (b)  the  number  introduced  into  the  body.  To  these  may 
be  added  (c)  the  path  of  infection. 

The  virulence,  i.e.  the  power  of  multiplying  in  the  body  and 
producing  disease,  varies  greatly  in  different  conditions,  and  the 
methods  by  which  it  can  be  diminished  or  increased  will  be 
afterwards  described  (vide  Chapter  XXI.).  One  important 
point  is  that  when  a  bacterium  has  been  enabled  to  invade 
and  multiply  in  the  tissues  of  an  animal,  its  virulence  for  that 
species  is  often  increased.  This  is  well  seen  in  the  case  of 
certain  bacteria  which  are  normally  present  on  the  skin  or 
mucous  surfaces.  Thus  it  has  been  repeatedly  proved  that  the 
bacillus  coli  cultivated  from  a  septic  peritonitis  is  much  more 
virulent  than  that  taken  from  the  bowel  of  the  same  animal. 
The  virulence  may  be  still  more  increased  by  inoculating  from 
one  animal  to  another  in  series — the  method  of  passage.  Widely 
different  effects  are,  of  course,  produced  on  the  virulence  being 
altered.  For  example,  a  streptococcus  which  produces  merely 
a  local  inflammation  or  suppuration,  may  produce  a  rapidly 
fatal  septicaemia  when  its  virulence  is  raised.  Virulence  also 
has  a  relation  to  the  animal  employed,  as  occasionally  on  being 
increased  for  one  species  of  animal  it  is  diminished  for  another. 
For  example,  streptococci,  on  being  inoculated  in  series  through 
a  number  of  mice,  acquire  increased  virulence  for  these  animals, 
but  become  less  virulent  for  rabbits  (Knorr).  The  theoretical 
consideration  of  virulence  must  be  reserved  for  a  later  chapter 
(see  Immunity). 

The  number  of  the  organisms  introduced,  i.e.  the  dose  of  the 
infecting  agent,  is  another  point  of  importance.  The  healthy 
tissues  can  usually  resist  a  certain  number  of  pathogenic 
organisms  of  given  virulence,  and  it  is  only  in  a  few  instances 
that  one  or  two  organisms  introduced  will  produce  a  fatal 
disease,  e.g.  the  case  of  anthrax  in  white  mice.  The  healthy 
peritoneum  of  a  rabbit  can  resist  and  destroy  a  considerable 
number  of  pyogenic  micrococci  without  any  serious  result,  but 


CONDITIONS  MODIFYING  PATHOGENICITY     177 

if  a  larger  dose  be  introduced,  a  fatal  peritonitis  may  follow. 
Again,  a  certain  quantity  of  a  particular  organism  injected 
subcutaneously  may  produce  only  a  local  inflammatory  change, 
but  in  the  case  of  a  larger  dose  the  organisms  may  gain  entrance 
to  the  blood  stream  and  produce  septicaemia.  There  is,  there- 
fore, for  a  particular  animal,  a  minimum  lethal  dose  which  can 
be  determined  by  experiment  only ;  a  dose,  moreover,  which  is 
modified  by  various  circumstances  difficult  to  control. 

The  path  of  infection  may  alter  the  result,  serious  effects  often 
following  a  direct  entrance  into  the  blood  stream.  Staphylo- 
cocci  injected  subcutaneously  in  a  rabbit  may  produce  only  a 
local  abscess,  whilst  on  intravenous  injection  multiple  abscesses 
in  certain  organs  may  result  and  death  may  follow.  Local 
inflammatory  reaction  with  subsequent  destruction  of  the 
organisms  may  be  restricted  to  the  site  of  infection  or  may 
occur  also  in  the  related  lymphatic  glands.  The  latter 
therefore  act  as  a  second  barrier  of  defence,  or  as  a  filtering 
mechanism  which  aids  in  protecting  against  blood  infection. 
This  is  well  illustrated  in  the  case  of  "poisoned  wounds."  In 
some  other  cases,  however,  the  organisms  are  very  rapidly 
destroyed  in  the  blood  stream,  and  Klemperer  has  found  that, 
in  the  dog,  subcutaneous  injection  of  the  pneumococcus  produces 
death  more  readily  than  intravenous  injection. 

'2.  The.  Xnhject  of  Infection. — Amongst  healthy  individuals 
susceptibility  and,  in  inverse  ratio,  resistance  to  a  particular 
microbe  may  vary  according  to  (a)  species,  (b)  race  .and  in- 
dividual peculiarities,  (c)  age.  Different  species  of  the  lower 
animals  show  the  widest  variation  in  this  respect,  some  being 
extremely  susceptible,  others  highly  resistant.  Then  there  are 
diseases,  such  as  leprosy,  gonorrhoea,  etc.,  which  appear  to  be 
peculiar  to  the  human  subject  and  have  not  yet  been  trans- 
mitted to  animals.  And  further,  there  are  others,  such  as 
cholera  and  typhoid,  which  do  not  naturally  affect  animals, 
and  the  typical  lesions  of  which  cannot  be  experimentally 
reproduced  in  them,  or  appear  only  imperfectly,  although 
pathogenic  effects  follow  inoculation  with  the  organisms.  In 
the  case  of  the  human  subject,  differences  in  susceptibility  to  a 
certain  disease  are  found  amongst  different  races,  and  also  amongst 
individuals  of  the  same  race,  as  is  well  seen  in  the  case  of  tubercle 
and  other  diseases.  Age  also  plays  an  important  part,  young 
subjects  being  more  liable  to  certain  diseases,  e.g.  to  diphtheria. 
Kurt  her.  at  different  periods  of  life  certain  parts  of  the  body  are 
more  susceptible,  for  example,  in  early  life,  the  bones  and  joints 
to  tubercular  and  acute  suppurative  affections. 

12 


178        RELATIONS  OF  BACTERIA  TO  DISEASE 

In  increasing  the  susceptibility  of  a  given  individual,  con- 
ditions of  local  or  general  diminished  vitality  play  the  most 
important  part.  It  has  been  experimentally  proved  that 
conditions  such  as  exposure  to  cold,  fatigue,  starvation,  etc., 
all  diminish  the  natural  resistance  to  bacterial  infection.  Rats 
naturally  immune  to  glanders  can  be  rendered  susceptible  by 
being  fed  with  phloridzin,  which  produces  a  sort  of  diabetes, 
a  large  amount  of  sugar  being  excreted  in  the  urine  (Leo). 
Guinea-pigs  may  resist  subcutaneous  injection  of  a  certain  dose 
of  the  typhoid  bacillus,  but  if  at  the  same  time  a  sterilised 
culture  of  the  bacillus  coli  be  injected  into  the  peritoneum,  they 
quickly  die  of  a  general  infection.  Also  a  local  susceptibility 
may  be  produced  by  injuring  or  diminishing  the  vitality  of  a 
part.  If,  for  example,  previous  to  an  intravenous  injection  of 
staphylococci,  the  aortic  cusps  of  a  rabbit  be  injured,  the 
organisms  may  settle  there  and  set  up  an  ulcerative  endocarditis ; 
or  if  a  bone  be  injured,  they  may  produce  suppuration  at  the 
part,  whereas  in  ordinary  circumstances  these  lesions  would  not 
take  place. 

Such  facts,  established  by  experiment  (and  many  others 
might  be  given),  illustrate  the  important  part  which  local  or 
general  conditions  of  diminished  vitality  may  play  in  the  pro- 
duction of  disease  in  the  human  subject.  This  has  long  been 
known  by  clinical  observation.  In  normal  conditions  the  blood 
and  tissues  of  the  body,  with  the  exception  of  the  skin  and 
certain  of  the  mucous  surfaces,  are  bacterium-free,  and  if  a  few 
organisms  gain  entrance,  they  are  destroyed.  But  if  the  vitality 
becomes  lowered,  their  entrance  becomes  easier  and  the  possibility 
of  their  multiplying  and  producing  disease  greatly  increased. 
In  this  way  the  favouring  part  played  by  fatigue,  cold,  etc., 
in  the  production  of  diseases  of  which  the  direct  cause  is  a 
bacterium,  may  be  understood.  It  is  important  to  keep  in  view 
in  this  connection  that  many  of  the  inflammation-producing 
and  pyogenic  organisms  .are  normally  present  on  the  skin  and 
various  mucous  surfaces.  The  action  of  a  certain  organism 
may  devitalise  the  tissues  to  such  an  extent  as  to  pave  the 
way  for  the  entrance  of  other  bacteria ;  we  may  mention  the 
liability  of  the  occurrence  of  pneumonia,  erysipelas,  and  various 
suppurative  conditions  in  the  course  of  or  following  infective 
fevers.  In  some  cases  the  specific  organism  may  produce  lesions 
through  which  the  other  organisms  gain  entrance,  e.g.  in  typhoid, 
diphtheria,  etc.  A  notable  example  of  diminished  resistance  to 
bacterial  infection  is  seen  in  the  case  of  diabetes ;  tuberculosis 
and  infection  with  pyogenic  organisms  are  prone  to  occur  in 


MODES  OF  BACTERIAL  ACTION  179 

this  disease,  and  are  of  a  severe  character.  It  is  not  uncommon 
to  find  in  the  bodies  of  those  who  have  died  from  chronic 
wasting  disease,  collections  of  uiicrococci  or  bacilli  in  the 
capillaries  of  various  organs,  which  have  entered  in  the  later 
hours  of  life;  that  is  to  say,  the  bacterium-free  condition  of 
the  blood  has  been  lust  in  the  period  of  prostration  preceding 
death. 

The  methods  by  which  the  natural  resistance  may  be  speci- 
fically increased  belong  to  the  subject  of  immunity,  and  are 
dc-MTibed  in  the  chapter  on  that  subject. 

Modes  of  Bacterial  Action. — In  the  production  of  disease  by 
micro-organisms  there  are  two  main  factors  involved,  namely, 
(a)  the  multiplication  of  the  living  organisms  after  they  have 
entered  tin-  body,  and  (6)  the  production  by  them  of  poisons 
\\hirh  may  act  both  upon  the  tissues  around  and  upon  the  body 
generally.  The  former  corresponds  to  infection,  the  latter  is  of 
the  nature  of  intoxication  or  poisoning.  In  different  diseases 
one  of  these  is  usually  the  more  prominent  feature,  but  both  are 
always  more  or  less  concerned. 

1.  Inflation  and  Distribution  of  the  Bacteria  in  the  Body. — 
After  pathogenic  bacteria  have  invaded  the  tissues,  or  in  other 
words,  after  infection  by  bacteria  has  taken  place,  their  further 
behaviour  varies  greatly  in  different  cases.  In  certain  cases 
they  may  reach  and  multiply  in  the  blood  stream,  producing  a 
fatal  septicaemia.  In  the  lower  animals  this  multiplication  of 
the  organisms  in  the  blood  throughout  the  body  may  be  very 
extensive  (for  example,  the  septicaemia 'produced  by  the  pneunio- 
coccus  in  rabbits) ;  but  in  septicaemia  in  man  it  very  seldom,  if 
ever,  occurs  to  so  great  a  degree,  the  organisms  rarely  remain  in 
large  numbers  in  the  circulating  blood,  and  their  detection  in  it 
during  life  by  microscopic  examination  is  rare,  and  even  culture 
methods  may  give  negative  results  unless  a  large  amount  of  blood 
is  used.  In  such  cases,  however,  the  organisms  may  be  found 
}><>.<t  mortem  lying  in  large  numbers  within  the  capillaries  of 
various  organs,  e.g.  in  cases  of  septicaemia  produced  by  strepto- 
cocci. In  the  human  subject  more  frequently  one  of  two  things 
happens.  In  the  first  place,  the  organisms  may  remain  local, 
producing  little  reaction  around  them,  as  in  tetanus,  or  a  well- 
marked  lesion,  as  in  diphtheria,  etc.  Or  in  the  second  place, 
they  may  pa<s  by  the  lymph  or  blood  stream  toother  parts  or 
organs  in  which  they  settle,  multiply,  and  produce  lesions,  as  in 
tubercle. 

L'.  /'/•'"///••//"//  of  CJwmical  Poisons. — In  all  these  cases  the 
growth  of  the  organisms  is  accompanied  by  the  formation  of 


180        RELATIONS  OF  BACTERIA  TO  DISEASE 

chemical  products,  which  act  generally  or  locally  in  varying  degree 
as  toxic  substances.  The  toxic  substances  become  diffused 
throughout  the  system,  and  their  effects  are  manifested  chiefly 
by  symptoms  such  as  the  occurrence  of  fever,  disturbances  of 
the  circulatory,  respiratory,  and  nervous  systems,  etc.  In  some 
cases  corresponding  changes  in  the  tissues  are  found,  for  example, 
the  changes  in  the  nervous  system  in  diphtheria,  to  be  afterwards 
described.  The  general  toxic  effects  may  be  so  slight  as  to  be 
of  no  importance,  as  in  the  case  of  a  local  suppuration ;  or  they 
may  be  very  intense,  as  in  tetanus;  or  again,  less  severe  but 
producing  cachexia  by  their  long  continuance,  as  in  tuberculosis. 

The  occurrence  of  local  tissue  changes  or  lesions  produced  in 
the  neighbourhood  of  the  bacteria,  as  already  mentioned,  is  one 
of  the  most  striking  results  of  bacterial  action,  but  these  also 
must  be  traced  to  chemical  substances  formed  in  or  around  the 
bacteria,  and  either  directly  or  through  the  medium  of  ferments. 
In  this  case  it  is  more  difficult  to  demonstrate  the  mode  of 
action,  for  in  the  tissues  the  chemical  products  are  formed  by 
the  bacteria  slowly,  continuously,  and  in  a  certain  degree  of 
concentration,  and  these  conditions  cannot  be  exactly  repro- 
duced by  experiment.  It  is  also  to  be  noted  that  more  than  one 
poison  may  be  produced  by  a  given  bacterium,  e.g.  the  tetanus 
bacillus  (p.  424).  Further,  it  is  very  doubtful  whether  all  the 
chemical  substances  formed  by  a  certain  bacillus  growing  in  the 
tissues  are  also  formed  by  it  in  cultures  outside  the  body  (vide 
p.  191).  The  separated  toxin  of  diphtheria,  like  various  vegetable 
and  animal  toxins,  however,  possesses  a  local  toxic  action  of 
very  intense  character,  evidenced  often  by  extensive  necrotic 
change. 

The  injection  of  large  quantities  of  many  different  pathogenic 
organisms  in  the  dead  condition  results  in  the  production  of  a 
local  inflammatory  change  which  may  be  followed  by  suppura- 
tion, this  effect  being  possibly  brought  about  by  certain  sub- 
stances in  the  bacterial  protoplasm  common  to  various  species, 
or  at  least  possessing  a  common  physiological  action  (Buchner 
and  others).  When  dead  tubercle  bacilli,  however,  are  intro- 
duced into  the  blood  stream,  nodules  do  result  in  certain  parts 
which  have  a  resemblance  to  ordinary  tubercles.  In  this  case 
the  bodies  of  the  bacilli  evidently  contain  a  highly  resistant  and 
slowly  acting  substance  which  gradually  diffuses  around  and 
produces  effects  (vide  Tuberculosis). 

Summary. — We  may  say,  then,  that  the  action  of  bacteria  as 
disease-producers,  as  in  fact  their  power  to  exist  and  multiply  in 
the  living  body,  depends  upon  the  chemical  products  formed 


TISSUE  CHANGES  PRODUCED  BY  BACTERIA     181 

directly  or  indirectly  by  them.  This  action  is  shown  by  tissue 
'•//'////AN  produced  in  the  vicinity  of  the  bacteria  or  throughout 
thr  system,  and  l»y  /o.r/V  *// mjitunis  of  great  variety  of  degree  and 
character. 

We  shall  first  consider  the  effects  of  bacteria  on  the  body 
generally,  and  afterwards  the  nature  of  the  chemical  products. 

KI-TKCTS  OF  BACTERIAL  ACTION. 

These  may  l»e  for  convenience  arranged  in  a  tabular  form  as 
follows  : 

A.  Tissue  Changes. 

(1)  Local  changes,   i.e.  changes  produced  in  the  neigh- 

bourhood of  the  bacteria. 

Position  (a)  At  primary  lesion. 
(6)  At  secondary  foci. 

Character  (a)  Tissue  reactions  \  Acute  or 

(b)  Degeneration  and  necrosis/  chronic. 

(2)  Produced  at  a  distance  from  the  bacteria,  directly  or 

indirectly,  by  the  absorption  of  toxins. 

(a)  In  special  tissues — 

(a)  as  the  result  of  damage,  e.g.  nerve  cells 
and  fibres,  secreting  cells,  vessel  walls,  or 

((3)  changes  of  a  reactive  nature  in  the  blood- 
forming  organs. 

(b)  General   anatomical   changes,    the   effects   of 

malnutrition  or  of  increased  waste. 

B.  Symptoms  and  Cfianyes  in  Metabolism. 

The  occurrence  of  fever,  of  errors  of  assimilation  and 
elimination,  etc. 

A.  Tissue  Changes  produced  by  Bacteria. — The  effects  of 
I 'arterial  action  are  so  various  as  to  include  almost  all  known 
pathological  changes.  However  varied  in  character,  they  may 
be  classified  under  two  main  headings :  (a)  those  of  a  degenera- 
tive or  necrotic  nature,  the  direct  result  of  damage ;  and  (b)  those 
of  reactive  nature,  defensive  or  reparative.  The  former  are  the 
r.\l  res<i<m  of  the  necessary  vulnerability  of  the  tissues,  the  latter 
of  protective  powers  evolved  for  the  benefit  of  the  organism.  In 
the  means  of  defence  both  leucocytes  and  the  fixed  cells  of  the 


182        RELATIONS  OF  BACTERIA  TO  DISEASE 

tissues   are   concerned.     Both  show   phagocytic    properties,    i.e. 
have  the  power  of  taking  up  bacteria  into  their  protoplasm.     The 
cells  are  guided  towards  the  focus  of  infection  by  chemiotaxis, 
and  thus  we  find  that  different  bacteria  attract  different  cells. 
The  most  rapid  and  abundant  supply  of  phagocytes  is  seen  in 
the  case  of  suppurative  conditions  where  the  neutrophile  leuco- 
cytes of  the  blood  are  chiefly  concerned.     When  the  local  lesion 
is  of  some  extent  there  is   usually  an   increase   of  these    cells 
in  the  bloo'd — a  neutrophile  leucocytosis.     And  further,  observa- 
tion  has   shown   that    associated   with    this    there    is    in    the 
bone-marrow  an  increased  number  of  the  mother-cells  of  these 
leucocytes — the  neutrophile    myelocytes.     The    passage   of   the 
neutrophile  leucocytes  from  the  marrow  into  the  blood,  with  the 
resulting  leucocytosis,  is  also    apparently  due   to  the    absorbed 
bacterial  toxins  acting  chemiotactically  on  the  marrow.     These 
facts  abundantly  show  that  the  means  of  defence  is  not  a  mere 
local  mechanism,   but  that   increased    proliferative    activity   in 
distant  tissues  is  called  into  play.     In  addition  to  direct  phago- 
cytosis by  these  leucocytes,  there  is  now  abundant  evidence  that 
an  important  function  is  the  production  in  the  body  of  bactericidal 
and    other   antagonistic  substances.     In    other    cases    the  cells 
chiefly  involved   are  the  mononuclear   hyaline    leucocytes,  and 
with  them  the  endothelial  cells,  e.g.  of  serous  membranes,  often 
play  an  important  part  in  the   defence ;  this  is   well  seen  in 
typhoid  fever,  where  the  specific  bacillus  appears  to  have  little 
or  no  action  on  the  neutrophile    leucocytes.     In   other  cases, 
again,    the  reaction    is    chiefly  on   the  part  of  the   connective 
cells,  though  their  proliferation  is  always  associated  with  some 
variety  of  leucocytic  infiltration  and  usually  also  with  the  forma- 
tion of  new  blood  vessels.     Such  a  connective  tissue  reaction 
occurs  especially  in  slow  infections  or  in  the  later  stages  of  an 
acute   infection.     The   tissue    changes   resulting   from    cellular 
activity  in  the  presence  of  bacterial  invasion  are  naturally  very 
varied, — examples  of  this  will  be  found  in  subsequent  chapters, — 
but  they  may  be  said  to  be  manifestations  of  the  two  funda- 
mental processes  of  (a)  increased  functional  activity — movement, 
phagocytosis,  secretion,  etc.— and  (b)  increased  formative  activity 
— cell   growth  and    division.     The   exudation    from   the   blood 
vessels  has  been  variously  interpreted.     There  is  no  doubt  that 
the  exudate  has  bactericidal  properties  and  also  acts  as  a  diluting 
agent,  but  it  must  still  be  held  as  uncertain  whether  the  process 
of  exudation  ought  to  be  regarded  as  primarily  defensive  or  as 
the  direct  result  of  damage  to  the  endothelium  of  the  vessels. 
It  may  also  be  pointed  out  that  the  various  changes  referred  to 


LOCAL  LESIONS  183 

are  none  of  them  peculiar  to  bacterial  invasion ;  they  are  examples 
of  the  general  laws  of  tissue  change  under  abnormal  conditions, 
and  they  can  all  be  reproduced  by  chemical  substances  in  solution 
or  in  a  particulate  state.  What  constitutes  their  special  feature 
is  their  progressive  or  spreading  nature,  due  to  the  bacterial 
multiplication. 

(1)  Local  Lesions. — In  some  diseases  the  lesion  has  a  special 
site;  for  example,  the  lesion  of  typhoid  fever  and,  to  a  less 
extent,  that  of  diphtheria.  In  other  cases  it  depends  entirely 
upon  the  point  of  entrance,  e.g.  malignant  pustule  and  the  con- 
ditions known  as  wound  infections.  In  others  again,  there  is  a 
special  tendency  for  certain  parts  to  be  affected,  as  the  upper 
parts  of  the  lungs  in  tubercle.  In  some  cases  the  site  has  a 
mechanical  explanation. 

When  organisms  gain  an  entrance  to  the  blood  from  a  primary 
lesion,  the  organs  specially  liable  to  be  affected  vary  greatly  in 
different  diseases.  Pyogenic  cocci  show  a  special  tendency  to 
settle  in  the  capillaries  of  the  kidneys  and  produce  miliary 
abscesses,  whilst  these  lesions  rarely  occur  in  the  spleen.  On 
the  other  hand,  the  nodules  in  disseminated  tubercle  or  glanders 
are  much  more  numerous  in  the  spleen  than  in  the  kidneys, 
which  in  the  latter  disease  are  usually  free  from  them.  The 
important  point  is  that  the  position  of  the  disseminated  lesions 
is  not  to  be  explained  by  a  mechanical  process,  such  as  embolism, 
but  depends  upon  a  special  relation  between  the  organisms  and 
the  tissues,  which  may  be  spoken  of  either  as  a  selective  power 
on  the  part  of  the  organisms  or  a  special  susceptibility  of  tissues, 
possibly  in  part  due  to  their  affording  to  the  organisms  more 
suitable  conditions  of  nutriment.  Even  in  the  case  of  the 
lesions  produced  by  dead  tubercle  bacilli,  a  certain  selective 
character  is  observed. 

Acute  Local  Lesions. — The  local  inflammatory  reaction  presents 
different  characters  in  different  conditions.  It  may  be  accom- 
panied by  abundant  fibrinous  exudation,  or  by  great  catarrh  (in 
the  case  of  an  epithelial  surface),  or  by  haemorrhage,  or  by 
redema ;  it  may  be  localised  or  spreading  in  character ;  it  may 
be  followed  by  suppuration,  and  may  lead  up  to  necrosis  of 
the  tissues  of  the  part,  a  good  example  of  the  latter  event 
being  found  in  a  boil.  Examples  will  be  given  in  subsequent 
chapters.  The  necrotic  or  degenerative  changes  affecting 
especially  the  more  highly  developed  elements  of  tissues  are 
chiefly  produced  by  the  direct  action  of  the  bacterial  poisons, 
though  aided  by  the  disturbances  of  nutrition  involved  in  the 
vascular  phenomena.  It  may  here  be  pointed  out  that  a  well- 


184        RELATIONS  OF  BACTERIA  TO  DISEASE 

marked  inflammatory  reaction  is  often  found  in  animals  which 
occupy  a  medium  position  in  the  scale  of  susceptibility,  and  that 
an  organism  which  causes  a  general  infection  in  a  certain  animal 
may  produce  only  a  local  inflammation  when  its  virulence  is 
lessened. 

Chronic  Local  Lesions. — In  a  considerable  number  of  diseases 
produced  by  bacteria  the  local  tissue  reaction  is  a  more  chronic 
process  than  those  described ;  there  is  less  vascular  disturbance 
and  a  greater  preponderance  of  the  proliferative  processes,  lead- 
ing to  new  formation  of  connective  tissue.  This  formation 
may  occur  in  foci  here  and  there,  so  that  nodules  result,  or  it  may 
be  more  diffuse.  Such  changes  especially  occur  in  the  diseases 
often  known  as  the  infective  granulomata,  of  which  tubercle, 
leprosy,  glanders,  actinomycosis,  syphilis,  etc.,  are  examples. 
A  hard-and-fast  line,  however,  cannot  be  drawn  between  such 
conditions  and  those  described  above  as  acute.  In  glanders,  for 
example,  especially  in  the  human  subject,  the  lesion  often 
approaches  very  nearly  to  an  acute  suppurative  change,  and 
sometimes  actually  is  of  this  nature.  Whilst  in  these  diseases 
the  fundamental  change  is  the  same — namely,  a  reaction  to  an 
irritant  of  minor  intensity — the  exact  structural  characters  and 
arrangement  vary  in  different  diseases.  In  some  cases  the 
disease  may  be  identified  by  the  histological  changes  alone,  but 
on  the  other  hand,  this  is  often  impossible. 

(2)  General  Lesions  produced  by  Toxins. — In  the  various  in- 
fective conditions  produced  by  bacteria,  changes  commonly 
occur  in  certain  organs  unassociated  with  the  presence  of  the 
bacteria ;  these  are  produced  by  the  action  of  bacterial  products 
circulating  in  the  blood.  Many  such  lesions  can  be  produced 
experimentally.  The  secreting  cells  of  various  organs,  especially 
the  kidney  and  liver,  are  specially  liable  to  change  of  this  kind. 
Cloudy  swelling,  which  may  be  followed  by  fatty  change  or 
by  actual  necrosis  with  granular  disintegration,  is  common. 
Hyaline  change  in  the  walls  of  arterioles  may  occur,  and  in 
certain  chronic  conditions  amyloid  change  is  brought  about  in 
a  similar  manner.  The  latter  has  been  produced  in  animals 
by  repeated  injections  of  the  staphylococcus  aureus.  Capillary 
haemorrhages  are  not  uncommon,  and  are  in  many  cases  due  to 
an  increased  permeability  of  the  vessel  walls,  aided  by  changes 
in  the  blood  plasma,  as  evidenced  sometimes  by  diminished 
coagulability.  Similar  haemorrhages  may  follow  the  injection  of 
some  bacterial  toxins,  e.g.  of  diphtheria,  and  also  of  vegetable 
poisons,  e.g.  ricin  and  abrin.  Skin  eruptions  occurring  in  the 
exanthemata  are  probably  produced  in  the  same  way,  though  in 


DISTURBANCES  OF  METABOLISM,  ETC.        185 

many  of  the«r  diseases  the  causal  organism  has  not  yet  been 
isolated.  \\  e  have,  however,  the  important  fact  that  corre- 
sponding skin  eruptions  may  be  produced  by  poisoning  with 
certain  drugs.  In  the  nervous  system  degenerative  changes 
have  been  found  in  diphtheria,  both  in  the  spinal  cord  and  in 
the  peripheral  nerves,  and  have  been  reproduced  experimentally 
by  the  products  of  the  diphtheria  bacilli.  There  is  also  experi- 
mental evidence  that  the  bacillus  coli  communis  and  the  strepto- 
coccus pyogenes  may,  by  means  of  their  products,  produce  areas 
of  softening  in  the  spinal  cord,  and  this  may  furnish  an  ex- 
planation of  some  of  the  lesions  found  clinically.  It  is  also 
p  »-ilile  that  some  serous  inflammations  may  be  produced  in  the 
same  way. 

B.  Disturbances  of  Metabolism,  etc. — It  will  easily  be 
realised  that  such  profound  tissue  changes  as  have  been  detailed 
cannot  occur  without  great  interference  with  the  normal  bodily 
metabolism.  General  malnutrition  and  cachexia  are  of  common 
occurrence,  and  it  is  a  striking  fact  found  by  experiment  that 
alter  injection  of  bacterial  products,  e.g.  of  the  diphtheria 
bacillus,  a  marked  loss  of  body  weight  often  occurs  which  may 
be  progressive,  leading  to  the  death  of  the  animal.  In  bacterial 
disease  assimilation  is  often  imperfect,  for  the  digestive  glands 
are  affected,  it  may  be,  by  actual  poisoning  by  bacterial  products,  it 
may  be  by  the  occurrence  of  fever,  and  excretion  is  interfered  with 
by  the  damage  done  to  the  excretory  cells.  But  of  all  the  changes 
in  metabolism  the  most  difficult  to  understand  is  the  occurrence 
of  that  interference  with  the  heat-regulating  mechanism  which 
results  in  fever.  The  degree  and  course  of  the  latter  vary, 
sometimes  conforming  to  a  more  or  less  definite  type,  where  the 
bacilli  are  selective  in  their  field  of  operation,  as  in  croupous 
pneumonia  or  typhoid,  sometimes  being  of  a  very  irregular  kind, 
especially  when  the  bacteria  from  time  to  time  invade  fresh 
areas  of  the  body,  as  in  pyaemic  affections.  The  main  point  of 
interest  regarding  the  development  of  fever  is  as  to  whether  it  is 
a  direct  effect  of  the  circulation  of  bacterial  toxins,  or  if  it  is  to 
be  looked  on  as  part  of  the  reaction  of  the  body  against  the 
irritant.  This  question  has  still  to  be  settled,  and  all  that  we 
can  do  is  to  adduce  certain  facts  bearing  on  it.  Thus  in  diph- 
theiia  and  tetanus,  where  toxic  action  leading  to  degeneration 
plays  such  an  important  part,  fever  may  be  a  very  subsidiary 
feature,  except  in  the  terminal  stage  of  the  latter  disease ;  and 
in  fact  in  diphtheria  profoundly  toxic  effects  may  be  produced 
with  little  or  no  interference  with  heat  regulation.  On  the 
other  hand,  in  bacterial  disease,  where  defensive  and  reparative 


186        EELATIONS  OF  BACTERIA  TO  DISEASE 

processes  predominate,  fever  is  rarely  absent,  and  it  is  nearly 
always  present  when  there  is  an  active  leucocytosis  going  on. 
In  this  connection  it  may  be  remarked  that  several  observers 
have  found  that,  when  a  relatively  small  amount  of  the  dead 
bodies  of  certain  bacteria  are  injected  into  an  animal,  fever 
occurs ;  while  the  injection  of  a  large  amount  of  the  same  is 
followed  by  subnormal  temperatures  and  rapidly  fatal  collapse. 
It  might  appear  as  if  this  indicated  that  the  occurrence  of  fever 
had  a  beneficial  effect,  but  this  is  one  of  the  points  at  issue. 
Certainly  such  an  effect  is  not  due  to  the  bacteria  being  unable 
to  multiply  at  the  higher  degrees  of  temperature  occurring 
in  fever,  for  this  has  been  shown  not  to  be  the  case.  Whether 
the  increase  of  bodily  temperature  indicates  the  occurrence  of 
changes  resulting  in  the  production  of  bactericidal  bodies,  etc., 
is  very  doubtful ;  a  production  of  antagonistic  substances  may 
be  effected  without  the  occurrence  of  fever  or  of  any  apparent 
disturbance  of  health.  If  we  consider  the  site  of  the  heat 
production  in  fever  we  again  are  in  difficulties.  It  might  appear 
as  if  the  tissue  destruction,  indicated  by  the  occurrence  of  fatty 
degeneration,  would  lead  to  heat  development,  but  frequently 
excessive  heat  production  with  increased  proteid  metabolism 
occurs  without  any  discoverable  changes  in  the  tissues ;  and 
further,  in  phosphorus  poisoning  there  is  little  fever  with  great 
tissue  destruction.  The  increased  work  performed  by  the  heart 
in  most  bacterial  infections  no  doubt  contributes  to  the  rise  of 
bodily  temperature.  But  we  must  bear  in  mind  that  in  fever 
there  is  more  than  mere  increase  of  heat  production — there  is 
also  a  diminished  loss  of  heat  from  interference  with  the  nervous 
mechanism  of  the  sweat  apparatus.  The  known  facts  would 
indicate  that  in  fever  there  is  a  factor  involving  the  nervous 
system  to  be  taken  into  account.  The  whole  subject  is  thus 
very  obscure. 

Symptoms. — Many  of  the  symptoms  occurring  in  bacterial 
infections  are  produced  by  the  histological  changes  mentioned, 
as  can  be  readily  understood  ;  whilst  in  the  case  of  others,  corre- 
sponding changes  have  not  yet  been  discovered.  Of  the  latter 
those  associated  with  fever,  with  its  disturbances  of  metabolism 
and  manifold  affections  of  the  various  systems,  are  the  most 
important.  The  nervous  system  is  especially  liable  to  be 
affected  —  convulsions,  spasms,  coma,  paralysis,  etc.,  being 
common.  The  symptoms  due  to  disturbance  or  abolition  of  the 
functions  of  secretory  glands  also  constitute  an  important  group, 
forming,  as  they  do,  a  striking  analogy  to  what  is  found  in  the 
action  of  various  drugs. 


THE  TOXINS  PRODUCED  BY  BACTERIA        187 

These  tissue  changes  and  symptoms  are  given  only  as  illus- 
trative examples,  and  the  list  might  easily  be  greatly  amplified. 
The  important  fact,  however,  is  that  nearly  all,  if  not  quite  all, 
tli>  changes  found  throughout  the  organs  (without  the  actual 
presence  of  bacteria),  and  also  the  symptoms  occurring  in  infec- 
tive diseases,  can  either  be  experimentally  reproduced  by  the  in- 
jection of  bacterial  poisons  or  have  an  analogy  in  the  action  of 
drugs. 

THE  TOXINS  PRODUCED  BY  BACTERIA. 

Early  Work  on  Toxins. — We  know  that  bacteria  are  capable 
of  giving  rise  to  poisonous  bodies  within  the  animal  body  and 
also  in  artificial  media.  We  know,  however,  comparatively  little 
of  the  actual  nature  of  such  bodies,  and  therefore  we  apply  to 
them  as  a  class  the  general  term  toxins.  The  .necessity  for 
accounting  for  the  general  pathogenic  effects  of  certain  bacteria, 
which  in  the  corresponding  diseases  were  not  distributed  through- 
out the  body,  directed  attention  to  the  probable  existence  of 
such  toxins ;  and  the  first  to  systematically  study  the  production 
of  such  poisonous  bodies  was  Brieger.  This  observer  isolated 
from  putrefying  substances,  and  also  from  bacterial  cultures, 
nitrogen-containing  bodies,  which  he  called  ptomaines.  Similar 
bodies  occurring  in  the  ordinary  metabolic  processes  of  the 
body  had  previously  been  described  and  called  leucomaines. 
I'toinaiii'.'s  isolated  from  pathogenic  bacteria  in  no  case  re- 
produced the  symptoms  of  the  disease,  except  perhaps  in  tetanus, 
and  this  only  owing  to  their  impurity.  The  methods  by  which 
they  were  isolated  were  faulty,  and  they  have  therefore  only  a 
historic  interest. 

The  introduction  of  the  principle  of  rendering  fluid  cultures 
bacteria-free  by  filtration  through  unglazed  porcelain,  and  its 
application  by  Roux  and  Yersin  to  obtain,  in  the  case  of  the 
1).  diphtherias,  a  solution  containing  a  toxin  which  reproduced 
the  symptoms  of  this  disease  (vide  Chapter  XVI.), -encouraged  the 
further  inquiry  as  to  the  nature  of  this  toxin.  An  attempt  on 
ihf  part  of  Brieger  and  Fraenkel'to  obtain  a  purified  diphtheria 
toxin  by  precipitating  bouillon  cultures  by  alcohol  (the  product 
being  denominated  a  toxalbumin)  did  not  greatly  advance 
knowledge  on  the  subject,  and  further  investigation  soon  showed 
that  specific  toxins  can  be  isolated  from  but  few  bacteria. 

General  Facts  regarding  Bacterial  Toxins. — The  following 
may  be  regarded  as  the  chief  facts  regarding  bacterial  toxins 
which  have  been  revealed  by  the  study,  partly  of  the  bodily 
tissues  of  animals  infected  by  the  bacteria  concerned,  partly  of 


188        THE  TOXINS  PRODUCED  BY  BACTERIA 

artificial  cultures  of  these  bacteria.  In  dealing  with  these  it  is 
necessary  to  distinguish  between  the  effects  produced  by  the 
actual  constituents  of  the  bacterial  protoplasm  (intracellular 
toxins)  and  those  which  in  a  few  bacteria  are  traceable  to 
soluble  substances  passing  out  into  the  media  in  which  these 
bacteria  may  be  growing  (extracellular  toxins).  The  former 
are  concerned  in  the  action  of  by  far  the  greater  number  of 
pathogenic  bacteria;  the  latter  account  for  the  pathogenic 
processes  originated  in  a  limited  number  of  cases  of  which 
diphtheria  and  tetanus  are  the  most  important.  This  dis- 
tinction is  important  as,  in  consequence  of  these  last  two 
diseases  having  had  much  attention  directed  towards  them  early 
in  the  history  of  research  on  the  subject,  there  has  hitherto 
been  too  much  tendency  to  take  for  granted  that  poisons  of 
a  similar  constitution  are  concerned  in  all  cases  of  bacterial 
intoxication.  At  present  such  an  assumption  is  not  justified 
by  facts,  and  we  do  not  even  know  whether  the  intracellular 
and  extracellular  toxins  belong  to  the  same  group  of  chemical 
bodies.  At  present,  however,  the  terms  are  used  as  a  con- 
venient means  of  accentuating  a  difference  in  solubility  between 
the  two  groups  of  toxic  bodies. 

The  dead  bodies  of  certain  bacteria  have  been  found  to  be 
very  toxic.  When,  for  instance,  tubercle  bacilli  are  killed  by 
heat  and  injected  into  the  body  tissues  of  a  susceptible  animal, 
tubercular  nodules  are  found  to  develop  round  the  sites  where 
they  have  lodged.  From  this  it  is  inferred  that  they  must  have 
contained  characteristic  toxins,  seeing  that  characteristic  lesions 
result.  The  bodies  of  such  organisms  as  the  pyogenic  cocci,  the 
b.  typhosus,  and  the  v.  cholerse  likewise  give  rise  to  pathogenic 
effects.  Such  intracellular  toxins  may  appear  in  the  fluids  in 
which  the  bacteria  are  living  (1)  by  excretion  in  an  unaltered 
or  altered  condition,  (2)  by  the  disintegration  of  the  bodies 
of  the  organisms  which  we  know  are  always  dying  in  any 
bacterial  growth.  The  death  of  bacteria  occurs  also  in  the 
body  of  an  infected  animal,  and  the  disintegration  of  these 
dead  bacteria  constitutes  an  important  means  by  which  the 
poisons  they  contain  are  absorbed.  There  is  some  evidence 
that  often  bacteria  originate  during  growth  poisons  which  are 
hurtful  to  their  own  vitality,  and  also  that  ferments  are  produced 
by  them  which  have  a  solvent  effect  on  the  poisoned  members 
of  the  colony.  Such  a  process  of  autolysis,  as  it  has  been  called, 
may  have  an  important  effect  in  liberating  intracellular  toxins. 
It  is  impossible,  at  present,  to  obtain  intracellular  toxins  apart 
from  other  derivatives  of  the  bacterial  protoplasm,  and  thus 


FACTS  REGARDING  BACTERIAL  TOXINS       189 

our  chief  knowledge  concerning  their  effects  is  derived  from  the 
study  of  what  happens  when  the  bodies  of  bacteria  killed  by 
chloroform  vapour  or  by  heat  are  injected  into  animals.  When 
effects  are  produced  by  such  injections  they  do  not  present  in 
any  particular  case  specific  characters.  They  are  of  the  nature 
•  •!'  u>'i irr;il  disturbances  of  metabolism,  as  manifested  by  fever, 
or  by  depression  of  temperature,  loss  of  weight,  etc.,  often  of 
such  serious  degree  as  to  result  in  death.  It  is  important  to 
note  that  when  pathogenic  effects  are  produced  these  usually 
appear  very  soon,  it  may  be  in  a  few  hours  after  injection  of  the 
toxic  material;  there  is  not  the  definite  period  of  incubation 
which  with  other  toxins  often  elapses  before  symptoms  appear. 

In  certain  cases  there  is  difficulty  in  understanding  the  action 
of  bacteria  which  neither  form  soluble  toxins  in  a  fluid  medium 
nor  possess  a  highly  toxic  protoplasm,  and  yet  with  which  we 
often  see  effects  produced  at  a  distance  from  the  focus  of 
infection,  e.g.  b.  anthracis.  To  explain  such  occurrences  it  has 
long  been  regarded  as  a  possibility  that  some  bacteria  are 
only  capable  of  producing  toxins  within  the  animal  tissues, 
and  it  has  further  been  thought  possible  that  bacteria,  such  as, 
for  example,  the  typhoid  bacillus,  which  do  distribute  into 
media  intracellular  toxins,  might  either  produce  these  toxins 
more  readily  in  the  tissues  or  might  produce  in  addition  other 
toxins  of  a  different  nature.  During  recent  years  such  toxins 
have  been  much  studied,  and  the  name  aygressins  has  been 
given  to  them.  The  evidence  adduced  for  the  existence  of 
these  aggressins  as  a  separate  group  of  bacterial  poisons  is  of 
the  following  kind :  An  animal  is  killed  by  a  dose  of  the 
typhoid,  dysentery,  cholera,  or  tubercle  bacillus,  or  by  a  staphy- 
lococcus,  the  organism  being  introduced  into  one  of  the  serous 
cavities.  After  death  the  serous  exudation,  which  in  all  these 
cases  is  present,  is  taken,  and  centrifugalised  to  remove  the 
bacteria  so  far  as  this  can  be  done  by  such  a  procedure ;  the 
bacteria  which  are  left  are  killed  by  shaking  the  fluid  up  with 
toluol  and  leaving  it  to  stand  for  some  days.  It  is  stated  that 
such  a  fluid  is  of  itself  without  pathogenic  effect,  but  has  the 
property  of  transforming  a  non-lethal  dose  of  the  bacterium  used 
into  one  having  fatal  effect.  Further,  the  effects  of  the  com- 
bined actions  of  the  bacteria  and  aggressins  are  often  of  a  much 
more  acute  character  than  can  be  obtained  with  toxic  products 
developed  in  vitro.  Thus,  in  the  case  of  the  action  of  a  non- 
lethal  dose  of  the  tubercle  bacillus  plus  its  aggressin,  it  is 
>;ii«  I  that  death  may  occur  in  twenty  hours,  a  result  never 
obtained  with  artificial  cultures  of  the  organism.  The  effects 


190       THE  TOXINS  PEODUCED  BY  BACTERIA 

produced  by  aggressins  are  attributed  to  a  paralysing  action 
on  the  phagocytic  functions  of  the  leucocytes.  The  subject  is 
full  of  difficulties,  and  in  the  case  of  certain  of  the  organisms 
employed  it  is  stated  that  results  similar  to  those  attributed  to 
aggressin  action  have  been  observed  with  macerated  cultures, — 
the  deduction  being  that  in  the  aggressins  we  are  merely  dealing 
with  concentrated  intracellular  toxins.  On  the  other  hand, 
as  evidence  of  the  existence  of  a  special  group  of  toxins,  it  has 
been  stated  that  a  special  type  of  immunity  against  the 
aggressins  can  be  originated.  Perhaps  the  most  important 
aspect  of  the  controversy  is  the  recognition  of  the  existence  of 
toxins  having  an  action  on  the  leucocytes.  A  poison  causing 
death  of  these  cells  in  connection  with  the  pus-forming  action  of 
the  pyogenic  cocci  has  been  described  under  the  name  of' 
leucocidin,  and  Eisenberg  records  that  in  in  vitro  mixtures  of 
leucocytes  and  cultures  of  the  bacillus  of  symptomatic  anthrax 
loss  of  inotility  and  degeneration  of  the  cells  may  be  observed. 
The  investigation  of  such  poisons  must  be  of  the  highest 
importance  in  view  of  the  part  played  by  the  blood-cells  in  the 
protection  of  the  body  against  infection,  and  it  is  possible  that 
toxins  having  a  fatal  effect  in  strong  concentrations  may,  when 
dilute,  be  responsible  for  the  phenomena  of  attraction  or  repulsion 
of  leucocytes  which  we  know  occur  round  a  focus  of  bacterial 
growth  in  the  body. 

Sometimes  the  media  in  which  bacteria  are  growing  become 
extremely  toxic.  This  is  more  marked  in  some  cases  than  in 
others.  The  two  best  examples  of  bacteria  thus  producing 
soluble  toxins  are  the  diphtheria  and  tetanus  bacilli.  In  these 
and  similar  cases  when  bouillon  cultures  are  filtered  bacterium- 
free  by  means  of  a  porcelain  filter,  toxic  fluids  are  obtained, 
which  on  injection  into  animals  reproduce  the  highly  character- 
istic symptoms  of  the  corresponding  diseases.  In  the  case  of 
the  b.  anthracis  and  of  many  others,  at  any  rate  when  growing 
in  artificial  media,  such  toxin  production  is  much  less  marked, 
a  filtered  bouillon  culture  being  relatively  non-toxic.  Poisons 
appearing  in  culture  media  have  been  called  extracellular  toxins, 
but  we  cannot  as  yet  say  whether  they  are  excreted  by  the 
bacteria  or  whether  they  are  produced  by  the  bacteria  acting  on 
the  constituents  of  the  media.  The  extracellular  toxins  are 
easily  obtainable  in  large  quantities,  and  it  is  their  nature  and 
effects  which  are  best  known.  No  method  has  been  discovered 
of  obtaining  them  in  a  pure  form,  and  our  knowledge  of  their 
properties  is  exclusively  derived  from  the  study  of  the  toxic 
nitrates  of  bouillon  cultures — these  filtrates  being  usually  re- 


FACTS  REGARDING  BACTERIAL  TOXINS      191 

ferrecl  to  simply  as  the  toxins.  These  toxins  differ  in  their 
ell'i.'ctN  from  the  intracellular  poisons  in  that  s^cific  actions  on 
certain  ti-> :i--s  are  often  manifested.  Thus  the  toxins  of  the 
diphtheria,  the  tetanus,  and  the  botulismus  bacilli  all  act  on 
the  ner\o  i-  system  ;  with  some  of  the  pyogenic  bacteria,  on  the 
other  haiul,  poisons,  probably  of  similar  nature,  produce  solution 
of  red  blood  corpuscles  (this  last  might  be  thought  to  explain 
the  aiuemias  so  common  in. the  associated  diseases,  but  it  is  to 
be  noted  that,  in  cultures  at  least,  these  htemolytic  toxins  are 
developed  in  very  small  amounts).  In  the  action  of  many  of 
these  toxins  the  occurrence  of  a  period  of  incubation  between 
the  introduction  of  the  poison  into  the  animal  tissues  and  the 
appearance  of  symptoms  is  often  a  feature. 

The  whole  question  of  the  parts  played  by  toxins  in  bacterial 
action  is  manifestly  very  complex.  On  the  one  hand,  we  have 
a  few  processes,  for  example,  diphtheria  and  tetanus,  in  which 
UTV  characteristic  effects  are  produced  on  special  tissues,  these 
being  accounted  for  by  the  formation  of  soluble  toxins  which 
are  capable  of  being  separated  from  the  bacterial  growths  in  vitro. 
On  the  other  hand,  we  have  the  great  mass  of  bacterial  infec- 
tions. With  regard  to  these,  the  distribution  of  the  bacteria 
in  the  bodies  of  infected  animals  makes  it  necessary  for  us 
to  take  for  granted  that  a  toxic  action  is  at  work.  All  that 
we  know,  however,  regarding  a  possible  explanation  of  such 
toxicity  is  that  the  bodies  of  the  bacteria  or  substances  directly 
derived  from  them  are  capable  of  producing  pathogenic  effects. 
These  effects  are  of  a  non-specific  character  in  the  sense  that 
they  are  not  the  result  of  an  action  on  any  particular  tissue  in 
the  body,  but  on  the  vital  processes  of  the  organism  as  a  whole. 
We  are  at  present  entirely  ignorant  of  the  interpretation  to  be 
put,  for  instance,  on  the  lowering  of  bodily  temperature  on  the 
one  hand  and  of  the  occurrence  of  fever  on  the  other,  both  of 
which  may  be  produced  by  the  injection  of  the  so-called  intra- 
cellular toxins  in  varying  doses,  and  we  are  ignorant  of  the 
relations  which  either  event  may  have  to  the  bringing  into  play 
of  the  defensive  mechanisms  of  the  body.  At  the  same  time  we 
must  admit  the  possibility  that  with  any  one  species  of  organism 
different  effects  may  be  produced  by,  it  may  be,  different  elements 
in  the  protoplasm  of  the  invading  bacterial  cell.  Some  of  these 
elements  may  act  on  certain  groups  of  specialised  cells  of  the 
body,  such  as  those  of  the  nervous  system,  liver,  or  kidneys, 
giving  rise  to  what  we  are  forced  to  describe  in  general  terms  as 
disturbances  of  metabolism.  Other  poisonous  elements  may 
mainly  act  on  the  defensive  cells  of  the  body,  of  which  the 


192       THE  TOXINS  PRODUCED  BY  BACTERIA 

leucocytes  may  be  taken  as  the  type.  Here  a  small  dose  of 
toxin  may  stimulate  these  cells  to  an  activity  which  results  in 
the  infection  being  thrown  off,  either  by  the  poison  being  neutra- 
lised, or  by  the  supply  of  toxin  being  cut  off  by  the  killing  of 
the  bacterium  producing  it.  A  large  dose  of  such  a  toxin,  may, 
on  the  other  hand,  altogether  break  down  the  defensive  mechanism 
of  the  invaded  body.  A  possible  complexity  in  toxic  action 
may  occur  even  in  such  an  apparently  simple  case  as  diphtheria. 
As  will  be  seen  later,  the  special  neuro-toxin  excreted  by  the 
diphtheria  bacillus  can  be  neutralised  by  an  antitoxic  substance, 
but  the  action  of  this  does  not  necessarily  cause  the  death  of 
the  bacteria  in  the  throat  whose  capacity  for  multiplication  may 
be  dependent  on  a  vital  activity  of  the  protoplasm  distinct 
from  neurotoxin  production,  and  therefore  requiring  another 
mechanism  for  its  neutralisation.  The  complexity  of  the  toxic 
process  is  also  illustrated  by  the  facts  known  regarding  the 
cholera  vibrio.  In  man,  this  organism  is  confined  in  its  habitat 
to  the  intestinal  tract,  and  its  serious  effects  are  attributed  to 
the  absorption  of  toxins  therefrom.  On  the  other  hand,  in 
animals,  not  susceptible  to  such  intestinal  infection,  death  can 
be  readily  produced  by  the  injection  intraperitoneally  of  a  com- 
paratively small  amount  of  dead  cholera  vibrios,  and  it  will  be 
seen  in  the  chapter  on  Cholera  that  the  possibility  has  to  be 
faced  of  the  toxins  acting  in  the  two  conditions  being  different. 
Thus  it  is  possible  that  the  toxic  element  in  an  organism  which 
enables  it  to  effect  its  initial  multiplication  in  or  on  the  tissues 
is  not  necessarily  bound  up  with  the  toxicity  which  is  respons- 
ible for  the  manifestation  of  specific  disease  effects.  This  is 
borne  out  by  the  work  of  Grassberger  and  Schattenfroh  on  the 
bacillus  of  symptomatic  anthrax.  In  this  case  an  organism, 
which  in  vitro  has  lost  to  a  large  extent  its  capacity  of  producing 
soluble  toxins,  may  show  great  capacity  for  multiplying  when 
introduced  into  a  susceptible  animal. 

There  is  another  point  which  must  be  kept  in  view,  namely, 
that  some  of  the  phenomena  which  have  been  regarded  as 
dependent  upon  the  activity  of  bacterial  toxins  may  possibly 
be  related  to  the  little-understood  process  of  anaphylaxis  (see 
Immunity).  Anaphylaxis  essentially  consists  in  the  develop- 
ment under  certain  circumstances  in  an  animal  of  a  hypersensi- 
tiveness  to  foreign  albuminous  materials  which  in  themselves 
are  not  essentially  toxic.  Effects  of  the  gravest  kind  may  be 
produced  during  this  period  of  hypersensitiveness,  and  it  has 
been  thought  that  some  of  the  phenomena  of  an  infectious 
disease,  e.g.  the  occurrence  of  an  incubation  period,  may  be 


THE  NATURE  OF  TOXINS  193 

accounted  for  by  the  development  of  hypersensitiveuess  to  the 
protoplasm  of  the  invading  bacteria.  It  may  be  said  here  that 
the  effect  seen  when  horse  serum  is  injected  into  a  rabbit 
i luring  its  hypersensitive  stage  to  this  substance  bears  a  striking 
resemblance  to  what  is  seen  in  natural  infection  in  man  by  the 
cholera  vibrio. 

The  phenomena  of  any  bacterial  disease  may  thus  in  reality 
be  due  to  very  different  and  complex  causes. 

The  Nature  of  Toxins. — There  is  still  comparatively  little 
known  regarding  this  subject,  and  it  chiefly  relates  to  the  extra- 
cellular toxins.  The  earlier  investigations  upon  toxins  suggested 
that  analogies  exist  between  the  modes  of  bacterial  action  and 
what  takes  place  in  ordinary  gastric  digestion,  and  the  idea  was 
worked  out  for  anthrax,  diphtheria,  tetanus,  and  ulcerative 
endocarditis  by  Sidney  Martin.  This  observer  found  that 
albumoses l  and  peptones  were  formed  by  the  action  of  the 
pathogenic  bacteria  studied,  and  further,  that  the  precipitate 
containing  these  albumoses  was  toxic.  A  similar  digestive 
action  has  been  traced  in  the  case  of  the  tubercle  bacillus  by 
Kiihne. 

Further  evidence  that  bacterial  toxins  are  either  albumoses 
or  bodies  having  a  still  smaller  molecule  is  adduced  by  C.  J. 
Martin.  This  worker,  by  filling  the  pores  of  a  Chamberland 
bougie  with  gelatin,  has  obtained  what  is  practically  a  strongly 
supported  colloid  membrane  through  which  dialysis  can  be  made 
to  take  place  under  great  pressure,  say,  of  compressed  oxygen. 
He  finds  that  in  such  an  apparatus  toxins — at  least  two  kinds 
tried — will  pass  through  just  as  an  albumose  will. 

Brieger  and  Boer,  working  with  bouillon  cultures  of  diphtheria 
and  tetanus,  separated,  by  precipitation  with  zinc  chloride, 

1  In  the  digestion  of  albumins  by  the  gastric  and  pancreatic  juices,  the 
albumoses  are  a  group  of  bodies  formed  preliminarily  to  the  production  of 
peptone.  Like  the  latter  they  differ  from  the  albumins  in  their  not  being 
coagulated  by  heat,  and  in  being  slightly  dialysable.  They  differ  from 
the  peptones  in  being  precipitated  by  dilute  acetic  acid  in  presence  of 
much  sodium  chloride,  and  also  by  neutral  saturated  sulphate  of  ammonia. 
Both  are  precipitated  by  alcohol.  The  first  albumoses  formed  in  digestion 
are  proto-alburnose  and  hetero-albumose,  which  ditfer  in  the  insolubility 
of  the  latter  in  hot  and  cold  water  (insolubility  and  coagulability  are 
ipiite  different  properties).  They  have  been  called  the  primary  albumoses. 
Hv  further  digestion  both  pass  into  the  secondary  albumose,  deutero- 
.illiumose,  which  differs  slightly  in  chemical  reactions  from  the  parent 
bodies,  e.g.  it  cannot  be  precipitated  from  watery  -solutions  by  saturated 
sodium  chloride  unless  a  trace  of  acetic  acid  be  present.  Dysalbumose  is 
probably  merely  a  temporary  modification  of  hetero-albumose.  Further 
digestion  of  deutero-albumose  results  in  the  formation  of  peptone. 

'3 


194        THE  TOXINS  PRODUCED  BY  BACTERIA 

bodies  which  show  characteristic  toxic  properties,  but  which  had 
the  reactions  neither  of  peptone,  albumose,  nor  albuminate,  and 
the  nature  of  which  is  unknown.  It  has  also  been  found  that 
the  bacteria  of  tubercle,  tetanus,  diphtheria,  and  ( cholera  can 
produce  toxins  when  growing  in  proteid-free  fluids  In  the  case 
of  diphtheria,  when  the  toxin  is  produced  in  such  a  fluid  a  proteid 
reaction  appears.  Of  course  this  need  not  necessarily  be  caused 
by  the  toxin.  Further  investigation  is  here  required,  for 
Uschinsky,  applying  Brieger  and  Boer's  method  to  a  toxin  so 
produced,  states  that  the  toxic  body  is  not  precipitated  by  zinc 
salts,  but  remains  free  in  the  medium.  If  the  toxins  are  really 
non-proteid  they  may,  on  the  one  hand,  be  the  final  product  of 
a  digestive  action,  or  they  may  be  the  manifestation  of  a  separate 
vital  activity  on  the  part  of  the  bacteria.  On  the  latter  theory 
the  toxicity  of  the  toxic  albumoses  of  Sidney  Martin  may  be  due  to 
the  precipitation  of  the  true  toxins  along  with  these  other  bodies. 
From  the  chemical  standpoint  this  is  quite  possible.  When  we 
take  into  account  the  extraordinary  potency  of  these  poisons  (in 
the  case  of  tetanus  the  fatal  dose  of  the  pure  poison  for  a 
guinea-pig  must  often  be  less  than  '00000 1  grm  ),  we  can  under- 
stand how  attempts  by  present  chemical  methods  to  isolate  them 
in  a  pure  condition  are  not  likely  to  be  successful,  and  of  their 
real  nature  we  know  nothing.  Friedberger  and  Moreschi  have 
shown  that  the  intravenous  injection  in  the  human  subject  of 
a  fraction  of  a  loopful  of  a  dead  typhoid  culture  gives  rise 
to  toxic  symptoms,  including  marked  febrile  reaction.  Such 
injections  are  followed  by  the  appearance  of  agglutinating  and 
bacteriolytic  substances  in  the  serum.  These  results  show  that 
intracellular  toxins  may  be  comparable  with  extracellular  toxins 
so  far  as  concerns  the  extremely  small  dose  sufficient  to  produce 
toxic  effects. 

Amongst  the  properties  of  the  extracellular  toxins  are 
the  following :  They  are  apparently  all  uncrystallisable  ;  they 
are  soluble  in  water  and  they  are  dialysable ;  they  are  pre- 
cipitated along  with  proteids  by  concentrated  alcohol,  and  also 
by  ammonium  sulphate;  if  they  are  proteids  they  are  either 
albumoses  or  allied  to  the  albumoses  ;  they  are  often  relatively  un- 
stable, having  their  toxicity  diminished  or  destroyed  by  heat  (the 
degree  of  heat  which  is  destructive  varies  much  in  different  cases), 
light,  and  by  certain  chemical  agents.  Their  potency  is  often 
altered  in  the  precipitations  practised  to  obtain  them  in  a  pure 
or  concentrated  condition,  but  among  the  precipitants  ammonium 
sulphate  has  little  if  any  harmful  effect.  Regarding  the  toxins 
which  are  more  intimately  associated  with  the  bacterial  proto- 


THE  NATURE  OF  TOXINS  195 

plasm  we  know  much  less,  but  it  is  probable  that,  chemically, 
their  nature  is  similar,  though  some  of  them  at  least  are  not  so 
ra-ily  injured  by  heat,  e.g.  those  of  the  tubercle  bacillus,  already 
mentioned.  In  the  case  of  all  toxins  the  fatal  dose  for  an 
animal  varies  with  the  species,  body  weight,  age,  and  previous 
conditions  as  to  food,  temperature,  etc.  In  estimating  the 
minimal  lethal  dose  of  a  toxin  these  factors  must  be  carefully 
considered. 

The  following  is  the  best  method  of  obtaining  concentrated  extra- 
cellular toxins  :  The  toxic  fluid  is  placed  in  a  shallow  dish,  and  ammonium 
sulphate  crystals  are  well  stirred  in  till  no  more  dissolve.  Fresh  crystals 
to  form  a  bulk  nearly  equal  to  that  of  the  whole  fluid  are  added,  and  the 
dish  set  in  an  incubator  at  37°  C.  overnight.  Next  day  a  brown  scum 
of  precipitate  will  be  found  floating  on  the  surface.  This  contains  the 
toxin.  It  is  skimmed  off  with  a  spoon,  placed  in  watch-glasses  ;  these 
are  dried  in  vocno  and  stored  in  the  dark,  also  in,  vacuo,  or  in  an  exsiccator 
<  nntaining  strong  sulphuric  acid.  For  use  the  contents  of  one  are 
dissolved  up  in  a  little  normal  saline  solution. 

The  comparison  of  the  action  of  bacteria  in  the  tissues  in 
the  production  of  these  toxins  to  what  takes  place  in  the  gastric 
digestion,,  has  raised  the  question  of  the  possibility  of  the  elabora- 
tion by  these  bacteria  of  ferments  by  which  the  process  may 
be  started.  Thus  Sidney  Martin  puts  forward  the  view  that 
ferments  may  be  produced  which  we  may  look  on  as  the 
primary  toxic  agents,  and  which  act  by  digesting  surrounding 
material  and  producing  albumoses — these  poisons  being,  as  it 
were,  secondary  poisons.  Hitherto  all  attempts  at  the  isolation 
of  bacterial  ferments  of  such  a  nature  have  failed. 

But  apart  from  the  fact  that  with  such  bacteria  as  those  of 
tetanus  and  diphtheria,  a  digestive  action  may  occur, analogies  have 
In-en  drawn  between  ferment  and  toxic  action.  The  chief  facts 
upon  which  such  analogies  have  been  founded  are  as  follows : 
Thus  the  toxic  products  of  these  and  other  bacteria  lose  their 
tuxicity  by  exposure  to  a  temperature  which  puts  an  end  to  the 
activity  of  such  an  undoubted  ferment  as  that  of  the  gastric 
juice.  If  a  bouillon  containing  diphtheria  toxin  be  heated  at 
G5°  C.  for  one  hour,  it  is  found  to  have  lost  much  of  its  toxic 
effect,  and  in  the  case  of  b.  tetani  all  the  toxicity  is  lost  by 
exposure  at  this  temperature.  In  both  diseases  there  is  a  still 
further  fact  which  is  adduced  in  favour  of  the  toxic  substances 
1  icing  of  the  nature  of  ferments,  namely,  the  existence  of  a 
driinitr  period  of  incubation  between  the  injection  of  the  toxic 
bodies  and  the  appearance  of  symptoms.  This  may  be  inter- 
preted as  showing  that  after  the  introduction  of,  say,  affiltered 


196       THE  TOXINS  PRODUCED  BY  BACTERIA 

bouillon  culture,  further  chemical  substances  are  formed  in  the 
body  before  the  actual  toxic  effect  is  produced.  Too  much 
reliance  must  not  be  placed  on  such  an  argument,  for  in  the 
case  of  tetanus,  at  least,  the  delay  may  be  explained  by  the  fact 
that  the  poison  apparently  has  to  travel  up  the  nerve  trunks 
before  the  real  poisonous  action  is  developed.  Further,  with 
some  poisons  presently  to  be  mentioned  which  are  closely  allied 
to  the  bacterial  toxins,  an '  incubation  period  may  not  exist. 
It  would  not  be  prudent  to  dogmatise  as  to  whether  the  toxins 
do  or  do  not  belong  to  such  an  ill-defined  group  of  substances 
as  the  ferments.  It  may  be  pointed  out,  however,  that  the 
essential  concept  of  a  ferment  is  that  of  a  body  which  can 
originate  change  without  itself  being  changed,  and  no  evidence 
has  been  adduced  that  toxins  fulfil  this  condition.  Another 
property  of  ferments  is  that  so  long  as  the  products  of  fermenta- 
tion are  removed,  the  action  of  a  given  amount  of  ferment  is 
indefinite.  Again,  in  the  case  of  toxins  no  evidence  of  such  an 
occurrence  has  been  found.  A  certain  amount  of  a  toxin  is 
always  associated  with  a  given  amount  of  disease  effect,  though 
a  process  of  elimination  of  waste  products  must  be  all  the  time 
going  on  in  the  animal's  body.  Again,  too  much  importance 
must  not  be  attached  to  loss  of  toxicity  by  toxins  at  relatively 
low  temperatures.  This  is  not  true  of  all  toxins,  and  further- 
more many  proteids  show  a  tendency  to  change  at  such 
temperatures ;  for  instance,  if  egg  albumin  be  kept  long 
enough  at  55°  C.  nearly  the  whole  of  it  will  be  coagulated. 
We  must  therefore  maintain  an  open  mind  on  this  subject. 

Similar  Vegetable  and  Animal  Poisons. — It  has  been  found  that 
the  bacterial  poisons  belong  to  a  group  of  toxic  bodies  all  present- 
ing very  similar  properties,  other  members  of  which  occur  widely 
in  the  vegetable  and  animal  kingdoms.  Among  plants  the  best- 
known  examples  are  the  ricin  and  abrin  poisons  obtained  by  making 
watery  emulsions  of  the  seeds  of  the  Ricinus  communis  and  the  Abrus 
precatorius  (jequirity)  respectively.  From  the  JRobinia  pseudacacia 
another  poison — robin — belonging  to  the  same  group  is  obtained.  The 
chemical  reactions  of  ricin  and  abrin  correspond  to  those  of  the  bacterial 
toxins.  They  are  soluble  in  water,  they  are  precipitable  by  alcohol,  but 
being  less  easily  dialysable  than  the  albumoses  they  have  been  called 
toxalbumins.  Their  toxicity  is  seriously  impaired  by  boiling,  and  they 
also  gradually  become  less  toxic  on  being  kept.  Both  are  among  the 
most  active  poisons  known — ricin  being  the  more  powerful.  When  they 
are  injected  subcutaneously  a  period  of  twenty-four  hours  usually  elapses 
— whatever  be  the  dose — before  symptoms  set  in.  Both  tend  to  produce 
great  inflammation  at  the  seat  of  inoculation,  which  in  the  case  of  ricin 
may  end  in  an  acute  necrosis  ;  in  fatal  cases  hsemorrhagic  enteritis  and 
nephritis  may  be  found.  Both  act  as  irritants  to  mucous  membranes, 
abrin  especially  being  capable  of  setting  up  most  acute  conjunctivitis. 


VEGETABLE  AND  ANIMAL  TOXINS  197 

In  the  actiou  of  a  poisonous  fungus,  Amanita  phalloides,  a  similar 
toxin  is  at  work.  After  an  incubation  period  of  some  hours,  symptoms  of 
abdominal  pain,  diarrhoea  with  bloody  stools,  and  later  jaundice  occur.  In 
vitro  the  toxin  has  a  haemolytic  action.  Like  other  poisons  of  this  class, 
an  antitoxin  can  be  produced  towards  the  fungus  poison. 

It  is  also  certain  that  the  poisons  of  scorpions  and  of  poisonous  snakes 
belong  to  the  same  group.  The  poisons  derived  from  the  latter  are 
usually  called  venins,  ana  a  very  representative  group  of  such  venins 
derived  from  different  species  has  been  studied.  To  speak  generally, 
there  is  derivable  from  the  natural  secretions  of  the  poison  glands  a 
series  of  venins  which  have  all  the  reactions  of  the  bodies  previously 
considered.  Like  ricin  and  abrin,  they  are  not  so  easily  dialysable  as 
bacterial  toxins,  and  therefore  have  also  been  classed  as  toxalbumins. 
Their  properties  are  also  similar  ;  many  of  them  are  destroyed  by  heat, 
but  the  degree  necessary  here  also  varies  much,  and  some  will  stand 
boiling.  There  is  also  evidence  that  in  a  crude  venin  there  may  be  several 
poisons  differently  sensitive  to  heat.  All  the  venius  are  very  powerful 
poisons,  but  here  there  is  practically  no  period  of  incubation — the  effects 
are  almost  immediate.  An  outstanding  feature  of  the  venins  is  the 
complexity  of  the  crude  poison  secreted  by  any  particular  species  of 
snake.  C.  J.  Martin,  in  summing  up  the  results  of  many  observers,  has 
pointed  out  that  different  venoms  have  been  found  to  contain  one  or 
more  of  the  following  poisons :  a  neurotoxiu  acting  on  the  respiratory 
centre,  a  neurotoxin  acting  on  the  nerve-endings  in  muscle,  a  toxin 
causing  haemolysis,  toxins  acting  on  other  cells,  e.g.  the  endothelium  of 
blood  vessels  (this  from  its  effects  has  been  named  ha-morrhagin), 
leucocytes,  nerve-cells,  a  toxin  causing  thrombosis,  a  toxin  having  an 
opposite  effect  and  preventing  coagulation,  a  toxin  neutralising  the 
I'.K tericidal  qualities  of  the  body  fluids  and  thus  favouring  putrefaction, 
a  toxin  causing  agglutination  of  the  red  blood  corpuscles,  a  proteolytic 
ferment,  a  toxin  causing  systolic  standstill  of  the  excited  heart.  Any 
particular  venom  contains  a  mixture  in  varying  proportions  of  such 
toxins,  and  the  different  effects  produced  by  the  bites  of  different  snakes 
largely  depend  on  this  variability  of  composition.  The  neurotoxic,  the 
thrombotic,  and  the  haemolytic  toxins  are  very  important  constituents 
of  any  venom.  The  toxicity  of  different  venoms  varies  much,  and  no 
general  statement  can  be  made  with  regard  to  the  toxicity  of  different 
poisons  towards  man.  Lamb  has  calculated  that  the  fatal  dose  of  crude 
cobra  venom  for  man  is  probably  about  '015  of  a  gramme,  and  that 
if  such  a  snake  bites  with  full  glands  many  times  this  dose  would 
probably  be  injected,  but,  of  course,  the  amount  emitted  depends  largely 
on  the  period  which  has  elapsed  since  the  animal  last  emptied  its  glands. 
When  a  dose  of  a  venom  not  sufficient  to  cause  immediate  death  from 
general  effects  be  given,  very  rapid  and  widespread  necrosis  often  may 
occur  in  a  few  hours  round  the  site  of  inoculation. 

An  extremely  important  fact  was  discovered  by  Flexner  and  Noguchi, 
namely,  that  the  haemolytic  toxin  of  cobra  venom  in  certain  cases  has  no 
action  by  itself,  but  produces  rapid  solution  of  red  corpuscles  when  some 
normal  serum  is  added,  the  latter  containing  a  labile  complement-like 
body,  which  activates  the  venom.  In  this  there  is  a  close  analogy  to 
what  holds  in  the  case  of  a  haemolytic  serum  deprived  of  complement  by 
heat  at  55°  C.  (p.  130).  Kyes  and  Sachs  further  showed  that  in  addition 
to  serum-complement  a  substance  with  definitely  known  constitution, 
namely  lecithin,  had  the  property  of  activating  the  haemolytic  substance 
in  cobra  venom,  the  two  apparently  uniting  to  form  an  actively  toxic 


198       THE  TOXINS  PRODUCED  BY  BACTERIA 

substance.  So  far  no  example  of  the  activation  of  a  bacterial  toxin  is 
known,  but  the  results  mentioned  point  to  the  possibility  of  this  occurring 
in  some  cases  in  the  tissues  of  the  body. 

There  is  another  group  of  toxic  manifestations  which  present  some 
analogies  to  those  of  the  bacterial  toxins,  but  concerning  which  very  little 
is  known.  The  best  example  of  these  is  found  in  the  toxic  properties  of 
the  serum  of  the  eel.  If  a  small  quantity  of  such  serum,  say  '25  of  a  c.c., 
be  injected  into  a  rabbit  subcutaneously,  death  occurs  in  a  few  minutes. 
Although  nothing  is  known  of  the  substances  giving  rise  to  such  effects, 
the  phenomenon  is  to  be  considered  in  relation,  on  the  one  hand,  to 
the  action  of  bacterial  toxins,  and  on  the  other  to  the  phenomenon  of 
anaphylaxis.  (See  Chapter  on  Immunity.) 

The  Theory  of  Toxic  Action. — While  we  know  little  of  the 
chemical  nature  of  any  toxins,  we  may,  from  our  knowledge  of 
their  properties,  group  together  the  tetanus  and  diphtheria 
poisons,  ricin,  abrin,  snake  poisons,  and  scorpion  poisons. 
Besides  the  points  of  agreement  already  noted,  all  possess  the 
further  property  that,  as  will  'be  afterwards  described,  when 
introduced  into  the  bodies  of  susceptible  animals  they  stimu- 
late the  production  of  substances  called  antitoxins.  The 
nature  of  the  antagonism  between  toxin  and  antitoxin  will 
be  discussed  later.  Here,  to  explain  what  follows,  it  may  be 
stated  (1)  that  the  molecule  of  toxin  forms  directly  a  combina- 
tion with  the  molecule  of  antitoxin,  and  (2)  that  it  has  been 
shown  that  toxin  molecules  may  lose  much  of  their  toxic  power 
and  still  be  capable  of  uniting  with  exactly  the  same  proportion 
of  antitoxin  molecules.  From  these  and  other  circumstances 
Ehrlich  has  advanced  the  view  that  the  toxin  molecule  has  .a 
very  complicated  structure,  and  contains  two  atom  groups.  One 
of  these,  the  haptophorous  (aTrreu/,  to  bind  to),  is  that  by 
which  combination  takes  place  with  the  antitoxin  molecule,  and 
also  with  presumably  corresponding  molecules  naturally  existing 
in  the  tissues.  .  The  other  atom  group  he  calls  the  toxophorous, 
and  it  is  to  this  that  the  toxic  effects  are  due.  This  atom  group 
is  bound  to  the  cell  elements,  e.y.  the  nerve  cells  in  tetanus,  by 
the  haptophorous  group.  Ehrlich  explains  the  loss  of  toxicity 
which  with  time  occurs  in,  say,  diphtheria  toxin,  on  the  theory 
that  the  toxophorous  group  undergoes  disintegration.  And  if  we 
suppose  that  the  haptophorous  group  remains  unaffected  we  can 
then  understand  how  a  toxin  may  have  its  toxicity  diminished 
and  still  require  the  same  proportion  of  antitoxin  molecules  for 
its  neutralisation.  To  the  bodies  whose  toxophorous  atom 
groups  have  become  degenerated,  Ehrlich  gives  the  name  toxoids. 
The  theory  may  afford  an  explanation  of  what  has  been  sus- 
pected, namely,  that  in  some  instances  toxins  derived  from 


THE  THEORY  OF  TOXIC  ACTION  199 

different  sources  may  be  related  to  one  another.  For  example, 
Ehrlich  has  pointed  out  that  ricin  produces  in  a  susceptible 
animal  body  an  antitoxin  which  corresponds  almost  completely 
with  that  produced  by  another  vegetable  poison,  robin  (vide 
supra),  though  ricin  and  robin  are  certainly  different.  This  may 
be  explained  on  the  supposition  that  robin  is  a  toxoid  of 
ricin,  i.e.  their  haptophorous  groups  correspond,  while  their 
toxophorous  differ.  The  evidence  on  which  Ehrlich's  deductions 
are  based  is  of  a  very  weighty  character,  but  another  view  of 
toxic  action  is  that  the  relation  between  a  toxin  and  the  cell 
on  which  it  acts  is  an  example  of  the  physical  phenomenon  of 
adsorption.  The  whole  subject  will  be  again  referred  to  in  the 
chapter  on  Immunity. 

With  regard  to  the  intracellular  toxins  we  shall  see  it  is 
difficult  to  determine  whether  or  not  they  share  with  the  extra- 
cellular poisons  the  property  of  stimulating  antitoxin  formation, 
—if  they  do  not,  then  they  may  belong  to  an  entirely  different 
class  of  substances.  It  is  certain  that  a  tolerance  against  such 
poisons  is  difficult  to  establish  and  is  not  of  a  lasting  character. 
We  thus  cannot  say  what  the  mechanism  is  by  which  these 
poisons  act.  It  may  be  said  that  Macfadyen,  by  grinding  up 
typhoid  bacilli  frozen  by  liquid  air,  claimed  that  on  thawing  he 
obtained  the  intracellular  toxins  in  liquid  form,  and  he  further 
stated  that  by  using  this  fluid  he  could  immunise  animals  not 
only  against  the  toxins  but  also  against  the  living  bacteria. 

\\V  have  already  pointed  out  that  those  who  claim  for  the 
aggressins  a  special  character  hold  that  the  activity  of  these 
bodies  has  as  its  effect  the  interference  with  the  phagocytic 
functions  of  the  leucocytes.  They  also  hold  that  a  special  type 
of  immunity  can  be  developed  against  the  aggressins. 


CHAPTER   VII. 

INFLAMMATORY  AND  SUPPURATIVE  CONDITIONS. 

THIS  subject  is  an  exceedingly  wide  one,  and  embraces  a  great 
many  pathological  conditions  which  in  their  general  characters 
and  results  are  widely  different.  Thus,  in  addition  to  suppura- 
tion, various  inflammations,  ulcerative  endocarditis,  septicaemia 
and  pyaemia,  will  come  up  for  consideration.  With  regard  to 
these,  the  two  following  general  statements,  established  by 
bacteriological  research,  may  be  made  in  introducing  the  subject. 
In  the  first  place,  there  is  no  one  specific  organism  for  any  one 
of  these  conditions;  various  organisms  may  produce  them, 
and  not  infrequently  more  than  one  organism  may  be 
present.  In  the  second  place,  the  same  organism  may  produce 
widely  varying  results  under  different  circumstances, — at  one 
time  a  local  inflammation  or  abscess,  at  another  multiple  sup- 
purations or  a  general  septicaemia.  The  principles  on  which 
this  diversity  in  results  depends  have  already  been  explained 
(p.  177).  Furthermore,  there  are  conditions  like  acute  pneu- 
monia, epidemic  meningitis,  acute  rheumatism,  etc.,  which  have 
practically  the  character  of  specific  diseases,  and  yet  which,  as 
regards  their  essential  pathology,  belong  to  the  same  class. 
The  arrangement  followed  is  to  a  certain  extent  one  of 
convenience. 

It  may  be  wrell  to  emphasise  some  of  the  chief  points  in  the 
pathology  of  these  conditions.  In  suppuration  the  two  main 
phenomena  are — (a)  a  progressive  immigration  of  leucocytes, 
chiefly  of  the  polymorpho-nuclear  (neutrophile)  variety,  and 
(b)  a  liquefaction  or  digestion  of  the  supporting  elements  of  the 
tissue  along  with  necrosis  of  the  cells  of  the  part.  The  result 
is  that  the  tissue  affected  becomes  replaced  by  the  cream-like 
fluid  called  pus.  A  suppurative  inflammation  is  thus  to  be 
distinguished  on  the  one  hand  from  an  inflammation  without 
destruction  of  tissue,  and  on  the  other  from  necrosis  or  death 
en  masse,  where  the  tissue  is  not  liquefied,  and  leucocyte 

200 


NATURE  OF  SUPPURATION  201 

accumulation  may  be  slight.  When,  however,  suppuration  is 
taking  place  in  a  very  dense  fibrous  tissue,  liquefaction  may  be 
incomplete,  and  a  portion  of  dead  tissue  or  slough  may  remain 
in  the  centre,  as  is  the  case  in  boils.  In  the  case  of  suppuration 
in  a  serous  cavity  the  two  chief  factors  are  the  progressive 
leucocytic  accumulation  and  the  disappearance  of  any  fibrin 
which  may  be  present. 

Many  experiments  have  been  performed  to  determine  whether 
suppuration  can  be  produced  in  the  absence  of  micro-organisms 
by  various  chemical  substances,  such  as  croton  oil,  nitrate  of 
silver,  turpentine,  etc. — care,  of  course,  being  taken  to  ensure 
the  absence  of  bacteria.  The  general  result  obtained  by  inde- 
I>endent  observers  is  that  as  a  rule  suppuration  does  not  follow, 
but  that  in  certain  animals  and  with  certain  substances  it  may, 
the  pus  being  free  from  bacteria.  Buchner  showed  that  sup- 
puration may  be  produced  by  the  injection  of  dead  bacteria,  e.g. 
sterilised  cultures  of  bacillus  pyocyaneus,  etc.  The  subject  has 
now  more  a  scientific  than  a  practical  interest,  and  the  general 
statement  may  be  made  that  practically  all  cases  of  true  sup- 
puration met  with  clinically  are  due  to  the  action  of  living 
micro-organisms. 

The  term  septicaemia,  is  applied  to  conditions  in  which  the 
organisms  multiply  within  the  blood  and  give  rise  to  symptoms 
of  general  poisoning,  without,  however,  producing  abscesses  in 
the  organs.  The  organisms  are  usually  more  numerous  in  the 
capillaries  of  internal  organs  than  in  the  peripheral  circulation, 
but  the  application  of  the  newer  methods  of  cultivation  has 
shown  that  they  can  be  detected  in  the  peripheral  blood  much 
more  frequently  than  was  formerly  supposed  to  be  the  case. 
The  essential  fact  in  pycemia,  on  the  other  hand,  is  the  occur- 
rence of  multiple  abscesses  in  internal  organs  and  other  parts  of 
the  body.  In  most  of  the  cases  of  typical  pyaemia,  common  in 
pre-antiseptic  days,  the  starting-point  of  the  disease  was  a  septic 
wound  with  bacterial  invasion  of  a  vein  leading  to  thrombosis 
and  secondary  embolism.  Multiple  foci  of  suppuration  may  be 
produced,  however,  in  other  ways,  as  will  be  described  below 
(p.  213).  If  the  term  "pyaemia"  be  used  to  embrace  all  such 
conditions,  their  method  of  production  should  always  be  dis- 
tinguished. 

BACTERIA  AS  CAUSES  OF  INFLAMMATION  AND  SUPPURATION. 

A  considerable  number  of  species  of  bacteria  have  been  found 
in  acute  inflammatory  and  suppurative  conditions,  and  of  these 


202          INFLAMMATION  AND  SUPPURATION 

many  have  been  proved  to  be  causally  related,  whilst  of  some 
others  the  exact  action  has  not  yet  been  fully  determined. 

Ogston,  who  was  one  of  the  first  to  study  this  question  (in 
1881),  found  that  the  organisms  most  frequently  present  were 
micrococci,  of  which  some  were  arranged  irregularly  in  clusters 
(staphylococci),  whilst  others  formed  chains  (streptococci).  He 
found  that  the  former  were  more  common  in  circumscribed 
acute  abscesses,  the  latter  in  spreading  suppurative  conditions. 
Rosenbach  shortly  afterwards  (1884),  by  means  of  cultures, 
differentiated  several  varieties  of  micrococci,  to  which  he  gave 
the  following  special  names :  staphylococcus  pyogenes  aureus, 
staphylococcus  pyogenes  albus,  streptococcus  pyogenes,  micrococcus 
pyogenes  tennis.  Other  organisms  are  met  with  in  suppuration, 
such  as  staphylococcus  pyogenes  citreus,  staphylococcus  cereus 
albus,  staphylococcus  cereus  Jlavus,  pneumococcus,  pneumobacillus, 
(Friedlander),  bacillus  pyogenes  foetidus  (Passet),  bacillus  coli 
communis,  bacillus  lactis  aerogenes,  bacillus  aerogenes  encapsul- 
atus,  bacillus  pyocyaneus,  micrococcus  tetragenus,  pneumococcus, 
pneumobacillus,  diplococcus  intracellularis  meningitidis,  and 
others.  Various  anaerobic  bacteria  are  also  concerned  in  the 
production  of  inflammation,  which  is  often  associated  with 
redema,  haemorrhage,  or  necrosis  (vide  Chap.  XVII.). 

In  secondary  inflammations  and  suppurations  following  acute 
diseases,  the  corresponding  organisms  have  been  found  in  some 
cases,  such  as  gonococcus,  typhoid  bacillus,  influenza  bacillus, 
etc.  Suppuration  is  also  produced  by  the  actinomyces  and  the 
glanders  bacillus,  and  sometimes  chronic  tubercular  lesions  have 
a  suppurative  character. 

Staphylococcus  Pyogenes  Aureus. — Microscopical  Characters. 
— This  organism  is  a  spherical  coccus  about  '9  //,  in  diameter, 
which  grows  irregularly  in  clusters  or  masses  (Fig.  50).  It 
stains  readily  with  all  the  basic  aniline  dyes,  and  retains  the 
colour  in  Gram's  method  (Plate  I.,  Fig.  1). 

Cultivation. — It  grows  readily  in  all  the  ordinary  media  at 
the  room  temperature,  though  much  more  rapidly  at  the 
temperature  of  the  body.  In  stab  cultures  in  peptone  gelatin 
a  streak  of  growth  is  visible  on  the  day  after  inoculation,  and 
on  the  second  or  third  day  liquefaction  commences  at  the  top. 
As  liquefaction  proceeds,  the  growth  falls  to  the  bottom  as  a 
flocculent  deposit,  which  soon  assumes  a  bright  yellow  colour, 
while  a  yellowish  film  may  form  on  the  surface,  the  fluid  portion 
still  remaining  turbid.  Ultimately  liquefaction  extends  out  to 
the  wall  of  the  tube  (Fig.  51).  In  gelatin  plates  colonies  may 
be  seen  with  the  low  power  of  the  microscope  in  twenty-four 


STAPHYLOCOCCnS  PYOGENES  AUREUS   203 


hours,  as  little  balls  somewhat  granular  on  the  surface  and  of 
brownish  colour.  On  the  second  day  they  are  visible  to  the 
naked  eye  as  whitish  yellow  points,  which  afterwards  become 


Ki(i.  r>0.-  Staphylococeoa  pyogenes  auveus, 
young  culture  on  agar,  showing  clumps 
of  cocci. 

Stained  with  weak  carbol-fuchsin.    x  1000. 


more  distinctly  yellow.  Liquefac- 
tion occurs  around  these,  and  little 
• -ii] is  are  formed,  at  the  bottom 
<>t  which  the  colonies  form  little 
yellowish  masses.  On  ayar,  a 
stroke  culture  forms  a  line  of 
abundant  yellowish  growth,  with 
smooth,  shining  surface,  well 
formed  after  twenty-four  hours  at 
37°  C.  Later  it  becomes  bright 
orange  in  colour,  and  resembles 
;i  -tivjik  of  oil  paint.  Single 
colonies  on  the  surface  of  agar  are  circular  discs  of  similar 
appearance,  which  may  reach  2  mm.  or  more  in  diameter. 
On  potatoes  it  grows  well  at  ordinary  temperature,  forming  a 
somewhat  abundant  layer  of  orange  colour.  In  bouillon  it 
produces  a  uniform  turbidity,  which  afterwards  settles  to  the 
bottom  as  an  abundant  layer  and  assumes  a  brownish  yellow 
tint.  In  tin-  various  media  it  renders  the  reaction  acid,  and  it 
n tabulates  milk,  in  which  it  readily  grows.  The  cultures  have 
a  siiiiii-wliat  sour  odour.  It  lia<  <-<>nsi<lrral>lr  tenacity  of  life 


FKI.  :")!.— Two  stab  cultures 
of  staphylococcus  pyogenes 
aureus  in  gelatin,  (a)  10  days 
old,  (6)  3  weeks  old,  showing 
liquefaction  of  the  medium 
and  characters  of  growth. 
Natural  size. 


204          INFLAMMATION  AND  SUPPURATION 

outside  the  body,  cultures  in  gelatin  often  being  alive  after 
having  been  kept  for  several  months. 

The  staphylococcus  pyogenes  albus  is  similar  in  character, 
with  the  exception  that  its  growth  on  all  the  media  is  white. 
The  colour  of  the  staphylococcus  aureus  may  become  less  dis- 
tinctly yellow  after  being  kept  for  some  time  in  culture,  but  it 
never  assumes  the  white  colour  of  the  staphylococcus  albus,  and 
it  has  not  been  found  possible  to  transform  the  one  organism 
into  the  other.  A  micrococcus  called  by  Welch  staphylococcus 
epidermidis  albus  is  practically  always  present  in  the  skin 
epithelium ;  it  is  distinguished  by  its  relatively  non-pathogenic 
properties  and  by  liquefying  gelatin  somewhat  slowly.  It  is 
probably  an  attenuated  variety  of  the  staphylococcus  albus. 

The  staphylococcus  pyogenes  citreus,  which  is  less  frequently 
met  with,  differs  in  the  colour  of  the  cultures,  being  a  lemon 
yellow,  and  is  less  virulent  than  the  other  two. 

The  staf)hylococcus  cereus  albus  and  staphylococcus  cere  us 
flavus  are  of  much  less  importance.  They  produce  a  wax-like 
growth  on  gelatin  without  liquefaction ;  hence  their  name. 

Streptococcus  pyogenes. — This  organism  (Plate  I.,  Fig.  1)  is  a 

coccus  of  slightly  larger 
size  than  the  staphylo- 
coccus  aureus,  about  1  /m 
in  diameter,  and  forms 
chains  which  may  contain 
a  large  number  of  mem- 
bers, especially  when  it  is 
growing  in  fluids  (Fig. 
52).  The  chains  vary 
somewhat  in  length  in 
different  specimens,  and 
on  this  ground  varieties 
have  been  distinguished, 
e.g.  the  streptococcus 
brevis  and  streptococcus 
longus  (vide  infra}.  As 
FIG.  52. -Streptococcus  pyogenes,  young  cul-  division  may  take  place 
ture  on  agar,  showing  chains  of  cocci.  in  many  of  the  COCci  in 

Stained  with  weak  carbol-fuchsiii.     x  1000.      a     cnajn     afc     the     same 

time,   the  appearance  of 

a  chain  of  diplococci  is  often  met  with.  In  young  cultures  the 
cocci  are  fairly  uniform  in  size,  but  after  a  time  they  present 
considerable  variations,  many  swelling  up  to  twice  their  normal 
diameter.  These  are  to  be  regarded  as  involution  forms.  In  its 


STREPTOCOCCUS  PYOGENES 


205 


staining  reactions  the  streptococcus  resembles  the  staphylococci 
described,  being  readily  coloured  by  Gram's  method. 

t 'ni 'ft' r,'  if  ion. — In  cultures  outside  the  body  the  streptococcus 
pyogenes  grows  much  more  slowly  than  the  staphylococci,  and 
also  «lies  out  more  readily,  being  in  every  respect  a  more  delicate 
organism. 

In  peptone  gelatin  a  stab  culture  shows,  about  the  second  day, 
a  thin  line,  which  in  its  subsequent  growth  is  seen  to  be  formed  of 
a  row  of  minute  rounded  colonies  of  whitish  colour,  which  may  be 
separate  at  the  lower  part  of  the 
puncture.  They  do  not  usually  ex- 
ceed the  size  of  a  small  pin's  head, 
this  size  being  reached  about  the  fifth 
or  sixth  day.  The  growth  does  not 
spread  on  the  surface,  and  no  lique- 
faction of  the  medium  occurs.  The 
colonies  in  gelatin  plates  have  a  cor- 
responding appearance,  being  minute 
>l>lierical  points  of  whitish  colour. 
A  somewhat  warm  temperature  is 
necessary  for  growth  ;  even  at  20°  C. 
-••me  varieties  do  not  grow.  On  the 
"//"/•  media,  growth  takes  place  along 
the  stroke  as  a  collection  of  small 
circular  discs  of  semi  -  translucent 
appearance,  which  show  a  great 
tendency  to  remain  separate  (Fig. 
53).  The  separate  colonies  remain 
sin; ill.  rarely  exceeding  1  mm.  in 
diameter.  Cultures  on  agar  kept  at 
the  body  temperature  may  often  be 
found  to  be  dead  after  ten  days.  On 
potato,  as  a  rule,  no  visible  growth 

takes  place.  In  milk  it  produces  a  strongly  acid  reaction  but  no 
••I'M ting  of  the  medium.  It  ferments  lactose,  saccharose,  and 
salicin  ( Andre wes  and  Horder) ;  it  produces  no  fermentation  of 
in ul in  in  Hiss's  serum- water-medium,  in  this  respect  differing 
from  the  pneumococcus.  It  has  a  strong  haemolytic  action,  as 
can  be  demonstrated  by  growing  it  in  blood-agar  plates  (p.  43). 
In  lioiiillon,  growth  forms  numerous  minute  granules  which  after- 
wards fall  to  the  bottom,  the  deposit,  which  is  usually  not  very 
abundant,  having  a  sandy  appearance.  The  api>earance  in 
broth,  however,  presents  variations  which  have  been  used  as  an 
aid  to  distinguish  different  species  of  streptococci.  It  has  been 


FIG.  53.— Culture  of  the 
streptococcus  pyogenes  on 
an  agar  plate,  showing 
numerous  colonies — three 
successive  strokes.  Twenty- 
four  hours'  growth.  Natu- 
ral size. 


206          INFLAMMATION  AND  SUPPURATION 

found  that  those  which  form  the  longest  chains  grow  most 
distinctly  in  the  form  of  spherical  granules,  those  forming  short 
chains  giving  rise  to  a  finer  deposit.  To  a  variety  which 
forms  distinct  spherules  of  minute  size  the  term  streptococcus 
conglomerate  has  been  given. 

Varieties  of  Streptococci. — Formerly  the  streptococcus  pyogenes 
and  the  streptococcus  erysipelatis  were  regarded  as  two  distinct 
species,  and  various  points  of  difference  between  them  were 
given.  Further  study,  and  especially  the  results  obtained  by 
modifying  the  virulence  (p.  210),  have  shown  that  these  dis- 
tinctions cannot  be  maintained,  and  now  practically  all  authorities 
are  agreed  that  the  two  organisms  are  one  and  the  same, 
erysipelas  being  produced  when  the  streptococcus  pyogenes  of  a 
certain  standard  of  virulence  gains  entrance  to  the  lymphatics  of 
the  skin.  Petruschky,  moreover,  showed  conclusively  by  inocu- 
lation that  a  streptococcus  cultivated  from  pus  could  cause 
erysipelas  in  the  human  subject. 

Streptococci  have  also  been  classified  according  to  the  length 
of  the  chains.  Thus  there  have  been  distinguished  (a)  strepto- 
coccus longus,  which  occurs  in  long  chains  and  is  pathogenic  to 
rabbits  and  mice;  (b)  streptococcus  brevis,  which  is  common  in 
the  mouth  in  normal  conditions,  and  is  usually  non-pathogenic ; 
and  (c)  streptococcus  conglomeratus,  so  called  from  its  forming  in 
bouillon  minute  granules  composed  of  very  long  chains.  It  may 
be  stated  that  pathogenic  streptococci  obtained  from  the  human 
subject  usually  form  fairly  long  chains  on  agar,  whilst  the  short 
streptococci  obtained  from  the  mouth  and  intestine  are  usually 
devoid  of  virulence.  But  to  these  statements  exceptions  occur, 
as  short  streptococci  may  be  associated  with  grave  lesions ;  it 
has  also  been  found  that  the  length  of  the  chains  is  not  a 
constant  feature. 

As  in  the  case  of  other  organisms  attempts  have  also  been  made  to 
differentiate  streptococci  by  means  of  tlieir  fermentative  properties. 
Mervyn  Gordon  introduced  for  this  purpose  nine  tests,  namely:  (1)  The 
clotting  of  milk,  (2)  the  reduction  of  neutral  red,  (3-9)  the  fermentation 
with  acid  production  of  saccharose,  lactose,  raffinose,  inulin,  salicin, 
coniferin,  and  mannite.  Andrewes  and  Horder  by  means  of  these  have 
differentiated  six  varieties,  of  which  five  occur  in  the  -human  subject. 
These  are  :  (a)  A  short-chained  form  called  streptococcus  mitis,  which 
occurs  chiefly  in  the  saliva  and  faeces  as  a  saprophyte.  It  ferments 
saccharose  and  lactose,  and  sometimes  the  glucosides  ;  it  produces  an  acid 
reaction  in  milk  but  no  clotting,  and  often  reduces  neutral-red.  (6)  The 
streptococcus  pyogenes,  which  is  the  most  important  pathogenic  variety,  and 
has  the  characters  described  above,  (c)  The  streptococcus  salivarius,  which 
corresponds  to  the  streptococcus  brevis  of  the  mouth,  and  which,  as 
regards  fermentative  action,  seems  to  bear  the  same  relation  to  the  next 


VARIETIES  OF  STREPTOCOCCI  207 

variety  as  the  streptococcus  mitis  does  to  the  streptococcus  pyogenes.  It 
ferments  saccharose,  lactose,  and  raffiuose,  sometimes  the  glucosides  and 
ruivly  inulin  ;  it  cl<>cs  milk  and  reduces  neutral-red.  (d>  Tlie  strepto- 
coccus iiiKjiiuHtHS,  wnicli  corresponds  with  the  so-called  streptococcus 
searlatime  and  the  streptococcus  conglomeratus.  It  ferments  saccharose 
and  lactose,  and  sometimes  ratfinose,  reduces  neutral-red,  and  is  actively 
hiumolytic.  It  us  .ally  clots  milk  and  does  not  grow  on  gelatin  at  20°  C. 
(e)  The  xti-i'iitiicuccns  fcecalis,  a.  short-chained  form,  which  abounds  in  the 
inte-tine  and  which  has  great  fermentative  activity,  and  reacts  positively 
t->  all  Gordon's  tests  with  the  exception  of  raffinose  and  inulin.  It  forms 
sulphuretted  i-ydropen,  and  is  devoid  «»f  haemolytic  action.  (/)  The 
sixth  variety  is  the  streptococcus  equiims.  which  is  common  in  the  air  and 
dust  of  t"wns,  and  appears  to  be  derived  from  horse  dung.1  It  fernu-nts 
saccharose  and  the  two  glucosides.  and  forms  little  or  no  ncid  in  milk.  It 
is,  ho\vev.-r,  to  be  noted  that  t>  all  these  varieties  variants  are  met  with. 
Schottmiiller  has  employed  the  appearance  of  the  colonies  of  strepto- 
cocci on  blood  agir  as  a  means  of  separating  varieties,  the  medium  used 
cou.-isting  of  i  wo  parts  human  blood  and  live  parts  melted  agar.  He 
distinguishes  the  streptoroccu*  lonyus  or  erysipclatis,  which  forms  grey 
colonies  and  has  a  marked  luemolytic  action  ;  a  streptococcus  mitior  or 
viridan-s,  a  short-chained  organism,  which  produces  small  green  colonies 
and  very  little  haemolysis  ;  and  a  streptococcus  muco  us  encapsulates, 
which,  as  its  name  indicates,  shows  wall-marked  capsules  and  produces 
colonies  which  have  a  slimy  consistence.  Mandelbaum  adds  to  these  the 
streptococcus  saprojthyticus,  which  is  without  haemoiytic  action.  It  should 
be  noted  that  on  blood  agar  the  pneumococcus  forms  green  colonies  and 
1  .K.I luces  little  «»r  no  haemolysis.  Levy  finds  that  a  2 '5  per  cent,  solu- 
ti  >n  of  taurocholate  of  sodium  in  bouillon  produces  complete  bacterio- 
lysis of  the  pneumococcus  and  the  streptococcus  mucosus,  while  it  has 
no  effect  on  other  varieties  of  streptococcus.  He  considers  the  strepto- 
coccus mucosus  to  lie  a  variety  of  pneumococcus.  The  general  statement 
may  be  made  that  most  of  the  streptococci  from  lesions  in  the  human 
subject  have  hsemolytic  action,  but  that  occasionally  streptococci  without 
this  property  are  found  even  in  severe  infections. 

It  will  be  thus  seen  from  this  account  that  the  streptococcus 
pyogenes  as  described  above  is  the  organism  most  frequently 
associated  with  the  pathogenic  processes,  and  that  short-chained 
forms  are  common  saprophytes  in  the.  human  body,  although 
they  may  be  associated  with  conditions  of  disease ;  these  may 
be  subdivided  according  to  their  fermentative  activity  as 
detailed.  And  lastly,  there  is  the  streptococcus  conglomeratus 
(anginosus),  which  is  specially  abundant  in  the  throat  in  scarlet 
fever,  though  it  also  occurs  in  other  acute  catarrhal  states.  No 
definite  statement  can  yet  be  made  as  to  the  etiological  relation 
of  streptococci  to  scarlet  fever  ;  we  can  only  say  that  streptococci 
are  almost  invariably  present  in  the  fauces,  and  that  to  them 
many  of  the  complications  of  the  disease  are  due. 

1  For  further  details,  reference  must  be  made  to  the  original  papers,  Lancet, 
September  1906,  ii.  708,  etc. 


208          INFLAMMATION  AND  SUPPURATION 

Bacillus  coli  communis.  —  The  microscopic  and  cultural  characters  are 
described  in  the  chapter  on  typhoid  fever.  The  bacillus  lactis  aerogenes 
and  the  bacillus  pyogenes  fo&tidus  closely  resemble  it  ;  they  are  either 
varieties  or  closely  related  species.  The  former  is  distinguished  by 
producing  more  abundant  gas  formation,  and  by  its  growth  on  gelatin, 
etc.,  being  thicker  and  whiter  than  that  of  the  bacillus  coli. 

Bacillus  aerogenes  encapsulatus  sometimes  invades  the  tissues  before 
death,  and  is  characterised  by  the  formation  of  bubbles  of  gas  in  the 
infected  parts.  Its  characters  are  described  in  Chapter  XVII. 

Bacillus  pyocyaneus.  —  This  organism  occurs  in  the  form  of  minute 
rods  1*5  to  3  ^  in  length  and  less  than  '5  ft  in  thickness  (Fig.  54). 

Occasionally  two   or   three 

_     ^  are  found  attached  end  to 

i  »  ss-  ^t  ,  end.      They    are    actively 

v   «A  ^   ,     .  N/  motile,  and    do    not    form 

•*  V%          V    ',  *  spores.     They  stain  readily 

£  *    ^     .      |>V*»'%*       •'  with    the     ordinary    basic 

^  A  A1     fc          /  *£  stains,  but  are  decolorised 

"    t      &  **  \     *\         •'•*•'        bJ  Gram's  method. 

'   *•  *    .    ,1$*  t         .*   ,*        t    '       •  Cultivation.  —  It    grows 

readily  on  all  the  ordinary 
media    at    the   room   tem- 
perature,  the  cultures  being 
distinguished    by   the    for- 
„»         mation  of  a  greenish  pig- 
•'<V  -         ment.       In    puncture   cul- 
*     """^     **.&        ^-  tures  in  peptone-gelatin  a 

;        '•  greyish     line     appears     in 

.£  -.•***  ^  *  twenty-four  hours,  and  at 

i  •        •*•  its  upper  part  a  small  cup 

*  of  liquefaction  forms  with- 

in  forty-eight    hours.     At 
FIG.  54.-Bacillus  pyocyaneus  ;  young          thig  time  a  slightlv  green. 


<f* 


gelatin.     The    liquefaction 

extends  pretty  rapidly,  the  fluid  portion  being  turbid  and  showing 
masses  of  growth  at  its  lower  part.  The  green  colour  becomes  more  and 
more  marked,  and  diffuses  through  the  gelatin.  Ultimately  liquefaction 
reaches  the  wall  of  the  tube.  In  plate  cultures  the  colonies  appear  as 
minute  whitish  points,  those  on  the  surface  being  the  larger.  Under  a 
low  power  of  the  microscope  they  have  a  brownish-yellow  colour  and 
show  a  nodulated  surface,  the  superficial  colonies  being  thinner  and 
larger.  Liquefaction  soon  occurs,  the  colonies  on  the  surface  forming 
shallow  cups  with  small  irregular  masses  of  growth  at  the  bottom,  the 
deep  colonies  small  spheres  of  liquefaction.  Around  the  colonies  a 
greenish  tint  appears.  On  agar  the  growth  forms  an  abundant  slimy 
greyish  layer  which  afterwards  becomes  greenish,  and  a  bright  green 
colour  diffuses  through  the  whole  substance  of  the  medium.  On  potatoes 
the  growth  is  an  abundant  reddish-brown  layer  resembling  that  of  the 
glanders  bacillus,  and  the  potato  sometimes  shows  a  greenish  discoloration. 
From  the  cultures  there  can  be  extracted  by  chloroform  a  coloured 
body,  pyocyanin,  which  belongs  to  the  aromatic  series,  and  crystallises 
in  the  form  of  long,  delicate  bluish-green  needles.  On  the  addition  of 
a  weak  acid  its  colour  changes  to  a  red. 


EXPERIMENTAL  INOCULATION  209 

This  organism  has  distinct  pathogenic  action  in  certain  animals. 
Subcutaneous  injection  of  small  doses  in  rabbits  may  produce  a  local 
suppuration,  but  if  the  dose  be  large,  spreading  haemorrhagic  oedema 
results,  which  may  be  attended  by  septicaemia.  Intravenous  injection 
may  produce,  according  to  the  dose,  rapid  septicaemia  with  nephritis,  or 
sometimes  a  more  chronic  condition  of  wasting  attended  by  alliuminuria. 

Micrococcus  tetragenus.  —  This  organism,  first  described  by  Galfky,  is 
characterised  by  the  fact  that  it  divides  in  two  planes  at  right  angles  to 
one  another  (Fig.  55),  and  is 
thus  generally  found  in  the 
tissues  in  groups  of  four,  or 
tetrads,  which  are  often  seen 
to  be  surrounded   by  a  cap- 
sule.     The  cocci  measure  1/4        f  ^|> 
in     diameter.      They     stain      J» 
readily  with  all  the  ordinary  C 

stains,  and  also  retain  tin.- 
stain  in  Gram's  method.  ^»  *  . 

It  grows  readily  on  all  the    I        ^  ^  1 

media  at  the  room  tempera-          f 
ture.     In  a  puncture  culture 

on   peptone-gelatin   a   pretty  J    «-. 

thick     whitish     line     forms  ^ 

along  the  track  of  the  needle,  «  ^ 

whilst  on  the  surface  there  >- 

is  a  thick   rounded   disc   of  *- 

whitish  colour.  The  gelatin 
is  not  liquefied.  On  the  sur- 
face of  a^ar  and  of  potato  *™-  5o.—  Micrococcus  tetragenus  ;  young 

culture  on  agar,  showing  tetrads. 
Staged  with  weatcarbol.f,,chsi,,     x  ,000. 


•« 
t 
£    . 


colour.      The  growth  on  all 

the  media  has  a  peculiar  viscid   or  tenacious  character,  owing  to  the 

gelatinous  character  of  the  sheaths  of  the  cocci. 

White  mice  are  exceedingly  susceptible  to  this  organism.  Subcutaneous 
injection  is  followed  by  a  general  septicaemia,  the  organism  being  found 
in  large  numbers  in  the  blood  throughout  the  body.  Guinea-pigs  are 
less  susceptible  ;  sometimes  only  a  local  abscess  with  a  good  deal  of 
necrotic  change  results  ;  sometimes  there  is  also  septicaemia. 

Experimental  Inoculation.  —  We  shall  consider  chiefly  the 
staphylococcus  pyogenes  aureus  and  the  streptococcus  pyogenes, 
an  these  have  been  most  fully  studii-d. 

It  may  be  stated  at  the  outset  that  the  occurrence  of  suppura- 
tion depends  upon  the  number  of  organisms  introduced  into  the 
tissues,  the  number  necessary  varying  not  only  in  different 
animals,  but  also  in  different  parts  of  the  same  animal,  —  a 
smaller  number  producing  suppuration  in  the  anterior  chamber 
of  the  eye,  for  example,  than  in  the  peritoneum.  The  virulence 
of  the  organism  also  may  vary,  and  corresponding  results  may 
be  produced.  Especially  is  this  so  in  the  case  of  the  strepto- 
coccus pyogenr-. 


210          INFLAMMATION  AND  SUPPURATION 

The  staphylococcus  aureus,  when  injected  subcutaneously  in 
suitable  numbers,  produces  an  acute  local  inflammation,  which 
is  followed  by  suppuration,  in  the  manner  described  above. 
If  a  large  dose  is  injected,  the  cocci  may  enter  the  blood  stream 
in  sufficient  numbers  to  cause  secondary  suppurative  foci  in 
internal  organs  (cf.  intravenous  injection). 

Intravenous  injection  in  rabbits,  for  example,  produces  in- 
teresting results,  which  vary  according  to  the  quantity  used.  If 
a  considerable  quantity  be  injected,  the  animal  may  die  in 
twenty-four  hours  of  a  general  septicaemia,  numerous  cocci  being 
present  in  the  capillaries  of  the  various  organs,  often  forming 
plugs.  If  a  smaller  quantity  be  used,  the  cocci  gradually  dis- 
appear from  the  circulating  blood;  some  become  destroyed, 
while  others  settle  in  the  capillary  walls  in  various  parts  and 
produce  minute  abscesses.  These  are  most  common  in  the 
kidneys,  where  they  occur  both  in  the  cortex  and  medulla  as 
minute  yellowish  areas  surrounded  by  a  zone  of  intense  con- 
gestion and  haemorrhage.  Similar  small  abscesses  may  be 
produced  in  the  heart  wall,  in  the  liver,  under  the  periosteum, 
and  in  the  interior  of  bones,  and  occasionally  in  the  striped 
muscles.  Very  rarely  indeed,  in  experimental  injection,  do  the 
cocci  settle  on  the  healthy  valves  of  the  heart.  If,  however, 
when  the  organisms  are  injected  into  the  blood,  there  be  any 
traumatism  of  a  valve,  or  of  any  other  part  of  the  body,  they 
show  a  special  tendency  to  settle  at  these  weakened  points. 

Experiments  on  the  human  subject  have  also  proved  the 
pyogenic  properties  of  those  organisms.  Garre  inoculated 
scratches  near  the  root  of  his  finger-nail  with  a  pure  culture,  a 
small  cutaneous  pustule  resulting;  and  by  rubbing  a  culture 
over  the  skin  of  the  forearm  he  caused  a  carbuncular  condition 
which  healed  only  after  some  weeks.  Confirmatory  experiments 
of  this  nature  were  made  by  Bockhart,  Bumm,  and  others. 

When  tested  experimentally,  the  staphylococcus  pyogenes 
albus  has  practically  the  same  pathogenic  effects  as  the  staphylo- 
coccus aureus. 

The  streptococcus  pyogenes  is  an  organism  the  virulence  of 
which  varies  much  according  to  the  diseased  condition  from 
which  it  has  been  obtained,  and  also  one  which  loses  its  virulence 
rapidly  in  cultures.  Even  highly  virulent  cultures,  if  grown 
under  ordinary  conditions,  in  the  course  of  time  lose  practically 
all  pathogenic  power.  By  passage  from  animal  to  animal,  how- 
ever, the  virulence  may  be  much  increased,  and  pari  passu  the 
effects  of  inoculation  are  correspondingly  varied.  Marmorek, 
for  example,  found  that  the  virulence  of  a  streptococcus  can  be 


BACILLUS  COLI  COMMUNIS  211 

enormously  increased  by  growing  it  alternately  (a)  in  a  mixture 
of  human  blood  serum  and  bouillon  (vide  p.  42),  and  (6)  in 
the  body  of  a  rabbit ;  ultimately,  after  several  passages  it  pos- 
sesses a  super-virulent  character,  so  that  even  an  extremely 
minute  dose  introduced  into  the  tissues  of  a  rabbit  produces 
rapid  septicaemia,  with  death  in  a  few  hours.  It  has  been 
proved  by  Marmorek's  experiments,  and  those  of  others,  that 
the  same  species  of  streptococcus  may  produce  at  one  time 
merely  a  passing  local  redness,  at  another  a  local  suppuration, 
at  another  a  spreading  erysipelatous  condition,  or  again  a 
general  septicsemic  infection,  according  as  its  virulence  is 
artificially  increased.  Such  experiments  are  of  extreme  import- 
ance as  explaining  to  some  extent  the  great  diversity  of  lesions 
in  the  human  subject  with  which  streptococci  are  associated. 

Bacillus  Coli  Communis. — The  virulence  of  this  organism 
also  varies  much,  and  can  be  increased  by  passage  from  animal 
to  animal.  Injection  into  the  serous  cavities  of  rabbits  pro- 
duces a  fibrinous  inflammation  which  becomes  purulent  if  the 
animal  lives  sufficiently  long.  If,  however,  the  virulence  of  the 
organism  be  of  a  high  order,  death  takes  place  before  suppura- 
tion is  established,  and  there  is  a  septicaemic  condition,  the 
organisms  occurring  in  large  numbers  in  the  blood.  Intravenous 
injection  of  a  few  drops  of  a  virulent  bouillon  culture  usually 
produces  a  rapid  septicaemia  with  scattered  haemorrhages  in 
various  organs. 

Lesions  in  the  Human  Subject. — The  following  statement 
may  be  made  with  regard  to  the  occurrence  of  the  chief  organisms 
mentioned,  in  the  various  suppurative  and  inflammatory  con- 
ditions in  the  human  subject.  The  account  is,  however,  by  no 
means  exhaustive,  as  clinical  bacteriology  has  shown  that  practi- 
cally every  part  of  the  body  may  be  the  site  of  a  lesion  produced 
by  the  pyogenic  bacteria.  It  may  also  be  noted  that  acute 
catarrhal  conditions  of  cavities  or  tubes  are  in  many  cases  also 
to  be  ascribed  to  their  presence. 

The  stapkylococci  are  the  most  common  causal  agents  in 
localised  abscesses,  in  pustules  on  the  skin,  in  carbuncles,  boils, 
etc.,  in  acute  suppurative  periostitis,  in  catarrhs  of  mucous 
surfaces,  in  ulcerative  endocarditis,  and  in  various  pyaemic 
conditions.  They  may  also  be  present  in  septicaemia. 

Streptococci  are  especially  found  in  spreading  inflammation 
with  or  without  suppuration ;  in  diffuse  phlegmonous  and 
erysipelatous  conditions,  suppurations  in  serous  membranes  and 
in  joints  (Fig.  56).  They  also  occur  in  acute  suppurative 
periostitis  and  ulcerative  endocarditis.  Secondary  abscesses  in 


212 


INFLAMMATION  AND  SUPPURATION 


FIG.  56. — Streptococci  in  acute  suppuration. 

Corrosive  film  ;  stained  by  Gram's  method 

and  safranin.     x  1000. 


lymphatic  glands  and  lymphangitis  are  also,  we  believe,  more 
frequently  caused  by  streptococci  than  staphylococci.  They  also 
produce  fibrinous  exudation  on  the  mucous  surfaces,  leading 

to  the  formation  of  false 
membrane  in  many  of  the 
cases  of  non-diphtheritic 
inflammation  of  the 
throat,  which  are  met 
with  in  scarlatina1  and 
other  conditions,  and  they 
are  also  the  organisms 
most  frequently  present 
in  acute  catarrhal  inflam- 
mations in  this  situation. 
In  puerperal  peritonitis 
they  are  frequently  found 
in  a  condition  of  purity, 
and  they  also  appear  to 
be  the  most  frequent 
cause  of  puerperal  septi- 
caemia, in  which  condition 
they  may  be  found  after 

death  in  the  capillaries  of  various  organs.  In  pyaemia  they  are 
frequently  present,  though  in  most  cases  associated  with  other 
pyogenic  organisms.  Some  cases  of  enteritis  in  infants — 
streptococcus  enteritis — are  also '  apparently  due  to  a  strepto- 
coccus, which,  however,  presents  in  cultures  certain  points  of 
difference  from  the  streptococcus  pyogenes. 

The  bacillus  coli  communis  is  found  in  a  great  many  inflam- 
matory and  suppurative  conditions  in  connection  with  the  ali- 
mentary tract — for  example,  in  suppuration  in  the  peritoneum, 
or  in  the  extra  peritoneal  tissue  with  or  without  perforation  of  the 
bowel,  in  the  peritonitis  following  strangulation  of  the  bowel,  in 
appendicitis  and  the  lesions  following  it,  in  suppuration  in  and 
around  the  bile  ducts,  etc.  It  may  ateo  occur  in  lesions  in  other 
parts  of  the  body, — endocarditis,  pleurisy,  etc.,  which  in  some 
cases  are  associated  with  lesions  of  the  intestine,  though  in  others 
such  cannot  be  found.  It  is  also  frequently  present  in  inflamma- 
tion of  the  urinary  passages,  cystitis,  pyelitis,  abscesses  in  the 
kidneys,  etc.,  these  lesions  being  in  fact  most  frequently  caused 
by  this  or  closely  allied  organisms. 

In  certain  cases  of  enteritis  it  is  probably  the  causal  agent, 

1  True  diphtheria  may  also  occasionally  be  associated  with  this  diseases 
usually  as  a  sequel. 


KN  TRANCE  AND  SPREAD  OF  BACTERIA      213 

though  tliis  is  difficult  of  proof,  as  it  is  much  increased  in 
numbers  in  practically  all  abnormal  conditions  of  the  intestine. 
We  may  ivmurk  that  it  has  been  repeatedly  proved  that  the 
bacillus  coli  cultivated  from  various  lesions  is  more  virulent  than 
that  in  the  intestine,  its  virulence  having  been  heightened  by 
growth  in  the  tissues. 

The  micrococcus  tetrayenus  is  often  found  in  suppurations  in 
the  region  of  the  mouth  or  in  the  neck,  and  also  occurs  in 
various  lesions  of  the  respiratory  tract,  in  phthisical  cavities, 
abscesses  in  the  lungs,  etc.  Sometimes  it  is  present  alone,  and 
probably  has  a  pyogenic  action  in  the  human  subject  under 
certain  conditions.  In  other  cases  it  is  associated  with  other 
organisms.  Cases  of  pyaemia  have  been  described  in  which  this 
organism  was  found  in  a  state  of  purity  in  the  pus  in  various 
situations.  In  this  latter  condition  the  pus  has  been  described 
as  possessing  an  oily,  viscous  character,  and  as  being  often 
blood-stained. 

The  bacillus  pyocyaneus  is  rarely  found  alone  in  pus,  though 
it  is  not  infrequent  along  with  other  organisms.  We  have  met 
with  it  several  times  in  cases  of  multiple  abscesses,  in  association 
with  the  staphylococcus  pyogenes  aureus.  Lately  some  diseases 
in  children  have  been  described  in  which  the  bacillus  pyocyaneus 
has  been  found  throughout  the  body ;  in  these  cases  the  chief 
symptoms  have  been  fever,  gastro-intestinal  irritation,  pustular 
or  petechial  eruptions  on  the  skin,  and  general  marasmus.  It 
has  also  been  said  to  be  constantly  present  in  pemphigus,  and 
it  certainly  occurs  in  some  cases  of  this  disease. 

Inflammatory  and  suppurative  conditions,  associated  with  the 
organisms  of  special  diseases,  will  be  described  in  the  respective 
chapters. 

Mode  of  Entrance  and  Spread. — Many  of  the  organisms  of 
suppuration  have  a  wide  distribution  in  nature,  and  many  also 
are  present  on  the  skin  and  mucous  membranes  of  healthy 
individuals.  Staphylococci  are  commonly  present  on  the  skin, 
and  also  occur  in  the  throat  and  other  parts,  and  streptococci 
can  often  be  cultivated  from  the  secretions  of  the  mouth  in 
normal  conditions.  The  pneumococcus  of  Fraenkel  and  the 
pneumobacillus  of  Friedlander  have  also  been  found  in  the 
mouth  and  in  the  nasal  cavity,  whilst  the  bacillus  coli  communis 
is  a  normal  inhabitant  of  the  intestinal  tract.  The  entrance  of 
these  organisms  into  the  deeper  tissues  when  a  surface  lesion 
occurs  can  be  readily  understood.  Their  action  will,  of  course, 
be  favoured  by  any  condition  of  depressed  vitality.  Though  in 
normal,  conditions  the  blood  is  bacterium-free,  we  must  suppose 


214 


INFLAMMATION  AND  SUPPURATION 


that  from  time  to  time  a  certain  number  of  such  organisms  gain 
entrance  to  it  from  trifling  lesions  of  the  skin  or  mucous  surfaces, 
the  possibilities  of  entrance  from  the  latter  being  especially 
numerous.  In  most  cases  they  are  killed  by  the  action  of  the 
healthy  serum  or  cells  of  the  body,  and  no  harm  results.  If, 
however,  there  be  a  local  weakness,  they  may  settle  in  that  part 
and  produce  suppuration,  and  from  this  other  parts  of  the  body 
may  be  infected.  Such  a  supposition  as  this  is  necessary  to 


FIG.  57. — Minute  focus  of  commencing  suppuration  in  brain— case 
of  acute  ulcerative  endocarditis.  In  the  centre  a  small  haemorrhage  ; 
to  right  side  dark  masses  of  staphylococci ;  zone  of  leucocytes  at 
periphery. 

Alum  carmine  and  Gram's  method,      x  50. 

explain  many  inflammatory  and  suppurative  conditions  met  with 
clinically.  In  some  cases  of  multiple  suppurations  due  to  staphy- 
lococcus  infection,  only  an  apparently  unimportant  surface  lesion 
is  present ;  whilst  in  others  no  lesion  can  be  found  to  explain 
the  origin  of  the  infection.  The  term  cryptogenetic  has  been 
applied  by  some  writers  to  such  cases  in  which  the  original  point 
of  infection  cannot  be  found,  but  its  use  is  scarcely  necessary. 

The  paths  of  secondary  infection  may  be  conveniently  sum- 
marised thus :  First,  by  lymphatics ;  in  this  way  the  lymphatic 


ENTRANCE  AND  SPREAD  OF  BACTERIA   215 

glands  may  be  infected,  and  also  serous  sacs  in  relation  to  the 
organs  where  the  primary  lesion  exists.  Second,  by  natural 
channels,  such  as  the  ureters  and  the  bile  ducts,  the  spread 
Itrin^  generally  associated  with  an  inflammatory  condition  of  the 
lining  i-pithrlium.  In  this  way  the  kidneys  and  liver  respectively 
may  be  infected.  Third,  by  the  blood  vessels :  (a)  by  a  few 
organisms  gaining  entrance  to  the  blood  from  a  local  lesion,  and 

v- 

. 


Kn:.  58.— Secondary  infection  of  a  glomerulus  of  kidney  by  the 
,  staphylococcus  aureus,  in   a  case  of  ulcerative  endocarditis.      The 
cocci  (stained  darkly)  are  seen  plugging  the  capillaries  and  also  lying 
free.      The  glomerulus  is  much  swollen,  infiltrated  by  leucocytes, 
and  partly  necrosed. 
Paraffin  section  ;  stained  by  Gram's  method  and  Bismarck-brown,    x  300. 

settling  in  a  favourable  nidus  or  a  damaged  tissue,  the  original 
path  of  infection  often  being  obscure;  (6)  by  a  septic  phlebitis 
with  suppurative  softening  of  the  thrombus  and  resulting  em- 
bolism ;  and  we  may  add  (c),  by  a  direct  extension  along  a  vein, 
producing  a  spreading  thrombosis  and  suppuration  within  the 
vein.  In  this  way  suppuration  may  spread  along  the  portal  vein 
to  the  liver  from  a  lesion  in  the  alimentary  canal,  the  condition 
being  known  as  pylephlebitis  suppurativa. 


216          INFLAMMATION  AND  SUPPURATION 

Although  many  of  the  lesions  produced  by  the  bacteria 
under  consideration  have  already  been  mentioned,  certain  con- 
ditions may  be  selected  for  further  consideration  on  account  of 
their  clinical  importance  or  bacteriological  interest. 

Endocarditis. — There  is  now  strong  presumptive  evidence 
that  all  cases  of  endocarditis  are  due  to  bacterial  infection.  In 
the  simple  or  vegetative  form,  so  often  the  result  of  acute 
rheumatism,  the  micrococcus  rheumaticus  (p.  221)  has  been 
cultivated  from  the  valves  in  a  certain  number  of  cases,  and  is 
probably  the  causal  agent  in  most  instances. 

Endocarditis  of  the  ulcerative  type  may  be  produced  by 
various  organisms,  chiefly  pyogenic.  Of  these  the  staphylococci 
and  streptococci  are  most  frequently  found.  In  some  cases  of 
ulcerative  endocarditis  following  pneumonia  the  pneumococcus 
(Fraenkel's)  is  present ;  in  these  the  vegetations  often  reach  a 
large  size  and  have  not  so  much  tendency  to  break  down  as  in 
the  case  of  staphylococcus  infections.  Other  organisms  have 
been  cultivated  from  different  cases  of  the  disease,  and  some  of 
these  have  received  special  names;  for  example,  the  diplo- 
coccus  endocarditis  encapsulatus,  bacillus  endocarditidis  griseus 
(Weichselbaum),  and  others.  In  some  cases  the  bacillus  coli 
communis  has  been  found,  and  occasionally  in  endocarditis 
following  typhoid  the  typhoid  bacillus  has  been  described  as  the 
organism  present,  but  further  observations  on  this  point  are 
desirable.  The  gonococcus  also  has  been  shown  to  affect  the 
heart  valves  (p.  256),  though  this  is  a  very  rare  occurrence. 
Tubercle  nodules  on  the  heart  valves  have  been  found  in  a  few 
cases  of  acute  tuberculosis,  though  no  vegetative  or  ulcerative 
condition  is  produced. 

In  some  cases,  though  we  believe  not  often,  the  organisms 
may  attack  healthy  valves,  producing  a  primary  ulcerative  endo- 
carditis, but  more  frequently  the  valves  have  been  the  seat*  of 
previous  endocarditis,  secondary  ulcerative  endocarditis  being 
thus  produced.  In  some  cases,  especially  when  the  valves  have 
been  previously  diseased,  the  source  of  the  infection  is  quite 
obscure.  It  is  evident  that  as  the  vegetations  are  composed  for 
the  most  part  of  unorganised  material,  they  do  not  offer  the 
same  resistance  to  the  growth  of  bacteria,  when  a  few  reach  them, 
as  a  healthy  cellular  tissue  does.  On  microscopic  examination 
of  the  diseased  valves  the  organisms  are  usually  to  be  found 
in  enormous  numbers  (Fig.  59).  By  their  action  breaking 
down  of  the  vegetations  occurs,  and  the  emboli  thus  produced 
act  as  the  carriers  of  infection  to  other  organs,  and  give  rise  to 
secondary  suppurations. 


PERIOSTITIS  AND  OSTEOMYELITIS  217 

••'iiftttal. — Occasionally  ulcerative  endocarditis  is  produced  by  the 
simple  intravenous  injection  of  staphylococci  aud  streptococci  into  the 
circulation,  but  this  is  a  very  rare  occurrence.  It  often  follows,  however, 
when  tlie  valves  have  heen  previously  injured.  Orth  and  Wyssokowitsch 
at  a  comparatively  early  date  produced  the  condition  by  damaging  the 
aortic  cusps  hy  a  glass  rod  introduced  through  the  carotid,  and  after- 
wards injecting  staphylococci  into  the  circulation.  Similar  experiments 
have  since  been  repeated  with  streptococci,  pneumococci,  and  other 
organisms,  with  like  result.  Ribbert  found  that  if  a  potato  culture  of 


PlO.  ~>9. — Section  of  a  vegetation  in  ulcerative  endocarditis  showing 
numerous  staphylococci  lying  in  the  spaces.  The  lower  portion  is  a 
tia-nient  in  process  of  separation. 

Stained  by  Gram's  method  and  Bismarck-brown,      x  600. 

the  staphylococcus  aureus  were  rubbed  down  in  salt  solution  so  as  to 
form  an  emulsion,  and  then  injected  into  the  circulation,  some  minute 
fragments  became  arrested  at  the  attachment  of  the  chordae  tendinere  and 
produced  an  ulcerative  endocarditis. 

Acute  Suppurative  Periostitis  and  Osteomyelitis. — Special 
mention  is  made  of  this  condition  on  account  of  its  comparative 
frequency  and  gravity.  The  great  majority  of  cases  are  caused 
by  the  pyogenic  cocci,  of  which  one  or  two  varieties  may  be 


218          INFLAMMATION  AND  SUPPURATION 

present,  the  staphylococcus  aureus,  however,  occurring  most 
frequently.  Pneumococci  have  been  found  alone  in  some  cases, 
and  in  a  considerable  number  of  cases  following  typhoid  fever 
the  bacillus  typhosus  has  been  found  alone.  In  others,  again, 
the  bacillus  coli  communis  is  present. 

The  affection  of  the  periosteum  or  interior  of  the  bones  by 
these  organisms,  which  is  especially  common  in  young  subjects, 
may  take  place  in  the  course  of  other  affections  produced  by 
the  same  organisms  or  in  the  course  of  infective  fevers,  but  in  a 
great  many  cases  the  path  of  entrance  cannot  be  determined. 
In  the  course  of  this  disease  serious  secondary  infections  are 
always  very  liable  to  follow,  such  as  small  abscesses  in  the 
kidneys,  heart- wall,  lungs,  liver,  etc.,  suppurations  in  serous 
cavities,  and  ulcerative  endocarditis ;  in  fact,  some  cases  present 
the  most  typical  examples  of  extreme  general  staphylococcus 
infection.  The  entrance  of  the  organisms  into  the  blood  stream 
from  the  lesion  of  the  bone  is  especially  favoured  by  the 
arrangement  of  the  veins  in  the  bone  and  marrow. 

Experimental. — Multiple  abscesses  in  the  bones  and  under  the  peri- 
osteum may  occur  in  simple  intravenous  injection  of  the  pyogenic  cocci 
into  the  blood,  and  are  especially  liable  to  be  formed  when  young 
animals  are  used.  These  abscesses  are  of  small  size,  and  do  not  spread 
in  the  same  way  as  in  the  natural  disease  in  the  human  subject. 

In  experiments  on  healthy  animals,  however,  the  conditions  are  not 
analogous  to  those  of  the  natural  disease.  We  must  presume  that  in  the 
latter  there  is  some  local  weakness  or  susceptibility  which  enables  the 
few  organisms  which  have  reached  the  part  by  the  blood  to  settle  and 
multiply.  Moreover,  if  a  bone  be  experimentally  injured,  e.g.  by  actual 
fracture  or  by  stripping  off  the  periosteum,  before  the  organisms  are 
injected,  then  a  much  more  extensive  suppuration  occurs  at  the  injured 
part. 

Erysipelas. — A  spreading  inflammatory  condition  of  the  skin 
may  be  produced  by  a  variety  of  organisms,  but  the  disease 
in  the  human  subject  in  its  characteristic  form  is  almost 
invariably  due  to  a  streptococcus,  as  was  shown  by  Fehleisen  in 
1884.  He  obtained  pure  cultures  of  the  organism,  and  gave  it 
the  name  of  streptococcus  erysipelatis ;  and,  further,  by  inocu- 
lations on  the  human  subject  as  a  therapeutic  measure  in 
malignant  disease,  he  was  able  to  reproduce  erysipelas.  As 
stated  above,  however,  one  after  another  of  the  supposed  points 
of  difference  between  the  streptococcus  of  erysipelas  and  that  of 
suppuration  has  broken  down,  and  it  is  now  generally  held  that 
erysipelas  is  produced  by  the  streptococcus  pyogenes  of  a  certain 
degree  of  virulence.  It  must  be  noted,  however,  that  erysipelas 
passes  from  patient  to  patient  as  erysipelas,  and  purulent  con- 


CONJUNCTIVITIS  219 

ditions  due  to  streptococci  do  not  appear  liable  to  be  followed 
by  erysipelas.  On  the  other  hand,  the  connection  between 
erysipelas  and  puerperal  septicaemia  is  well  established  clinically. 

In  a  case  of  erysipelas  the  streptococci  are  found  in  large 
numbers  in  the  lymphatics  of  the  cutis  and  underlying  tissues, 
just  beyond  the  swollen  margin  of  the  inflammatory  area.  As 
the  inflammation  advances  they  gradually  die  out,  and  after  a 
time  their  extension  at  the  periphery  comes  to  an  end.  The 
streptococci  may  extend  to  serous  and  synovial  cavities  and  set 
up  inflammatory  or  suppurative  change, — peritonitis,  meningitis, 
and  synovitis  may  thus  be  produced. 

Conjunctivitis. — A   considerable    number   of  organisms  are 
concerned  in  the  production  of  conjunctivitis  and  its  associated 
lesions.     Of  these  a  mini 
ber  appear  to   be  speci- 
ally associated  with  this 
region.      Thus    a    small 
organism,generally  known        /- 
as  the  Koch-Weeks  bacil-      / 
Ins,  is  the  most  common    / 
cause  of  acute  contagious    i 
conjunctivitis,    especially 
prevalent  in  Egypt,  but    • 
also     common     in      this 
country.     This  organism      V 
is     very    minute,    being 
little    more  than  1  /x  in 
length,  and  morphologic- 
ally   resembles    the     in- 

tinmr/.}    W-illiiQ  •   if«  ™n      FIG.  60.— Film  preparation  from  a  case  of 

acute  conjunctivitis,  showing  Koch- Weeks 

ditions  of  growth  are  bacilli,  chiefly  contained  within  a  leucocyte. 
even  more  restricted,  as  (From  a  preparation  by  Dr.  Inglis  Pollock.) 
it  rarely  grows  on  blood  x  1000< 

agar,    the    best    medium 

b-.-ing  serum  agar.  On  this  medium  it  produces  minute 
transparent  colonies  like  drops  of  dew.  The  obtaining  of  pure 
cultures  is  a  matter  of  considerable  difficulty,  and  it  is  nearly 
always  accompanied  by  the  xerosis  bacillus.  It  can  readily  be 
found  in  the  muco-purulent  secretion  by  staining  films  with 
weak  (1:10)  carbol-fuchsin,  and  is  often  to  be  seen  in  the  interior 
of  leucocytes  (Fig.  60).  Another  organism  exceedingly  like  the 
previous,  apparently  differing  from  it  only  in  the  rather  wider 
conditions  of  growth,  is  Miiller's  bacillus.  It  was  cultivated  by 
him  in  a  considerable  proportion  of  cases  of  trachoma,  but  its 


220 


INFLAMMATION  AND  SUPPURATION 


relation  to  this  condition  is  still  matter  of  dispute.  Another 
bacillus  which  is  now  well  recognised  is  the  diplo-bacillus  of 
conjunctivitis  first  described  by  Morax.  It  is  especially  common 
in  the  more  subacute  cases  of  conjunctivitis.  Eyre  found  it  in 
2*5  per  cent,  of  all  cases  of  conjunctivitis.  Its  cultural  characters 
are  given  below.  The  xerosis  bacillus,  which  is  a  small  diph- 
theroid  organism  (Fig.  119),  has  been  found  in  xerosis  of  the 
conjunctiva,  in  follicular  conjunctivitis,  and  in  other  conditions ; 
it  appears  to  occur  sometimes  also  in  the  normal  conjunctiva. 
It  is  doubtful  whether  it  has  any  pathogenic  action  of  importance. 
Acute  conjunctivitis  is  also  produced  by  the  pneumococcus, 
epidemics  of  the  disease  being  sometimes  due  to  this  organism, 
and  also  by  streptococci  and  staphylococci.  True  diphtheria  of 

the  conjunctiva  caused 
by  the  Klebs  -  Loffler 
bacillus  also  occurs, 
whilst  in  gonorrhoeal 
conjunctivitis,  often  of 
an  acute  purulent  type, 
the  gonococcus  is  pre- 
sent (p.  255). 

Diplo-bacillus  of  Con- 
junctivitis. —  This  organ- 
ism, discovered  by  Morax, 
is  a  small  plump  bacillus, 
measuring  1x2  /A,  and 
usually  occurring  in  pairs, 
or  in  short  chains  of  pairs 
(Fig.  61).  It  is  non-motile, 
does  not  form  spores,  and 
is  decolorised  by  Gram's 
method.  It  does  not  grow 
on  the  ordinary  gelatin  and 
agar  media,  the  addition 
of  blood  or  serum  being 
necessary.  On  serum  it  forms  small  rounded  colonies  which  produce 
small  pits  of  liquefaction ;  hence  it  has  been  called  the  bacillus 
lacunatus.  In  cultures  it  is  distinctly  pleomorphous,  and  involution 
forms  also  occur.  It  is  non-pathogenic  to  the  lower  animals. 

Acute  Rheumatism. — There  are  many  facts  which  point  to 
the  infective  nature  of  this  disease,  and  investigations  from  this 
point  of  view  have  yielded  important  results.  Of  the  organisms 
isolated,  the  one  which  appears  to  have  strongest  claims  is  a 
small  coccus  observed  by  Triboulet,  and  by  Westphal  and 
Wassermann,  the  characters  and  action  of  which  were  first 
investigated  in  this  country  by  Poynton  and  Paine.  It  is  now 


™       r.-,      T,-VI  ,.  .     , 

JIG.  61.— Film   preparation    of  conjunctival 

secretion,  showing  the  Morax  diplo-bacilhis 
of  conjunctivitis,      x  1000. 


ACUTE  RHEUMATISM  221 

usually  spoken  of  as  the  micrococcus  rhewmaticus.  The  organism 
is  sometimes  spoken  of  as  a  diplococcus,  but  it  is  best  described 
as  a  streptococcus  growing  in  short  chains ;  in  the  tissues,  how- 
ever, it  usually  occurs  in  pairs.  It  is  rather  smaller  than  the 
streptococcus  pyogenes,  and  although  it  can  be  stained  by  Gram's 
method,  it  loses  the  colour  more  readily  than  the  streptococcus. 
In  the  various  media  it  produces  a  large  amount  of  acid,  and 
usually  clots  milk  after  incubation  for  two  days;  on  blood  agar 
it  alters  the  haemoglobin  to  a  brownish  colour.  Its  growth  on 
media  generally  is  more  luxuriant  than  that  of  the  strepto- 
coccus, and  it  grows  well  on  gelatin  at  20°  C.  Injection 
of  pure  cultures  in  rabbits  often  produces  polyarthritis  and 
synovitis,  valvulitis  and  pericarditis,  without  any  suppurative 
change — lesions  which  it  is  stated  are  not  produced  by  the 
ordinary  streptococci  (Beattie).  In  one  or  two  instances 
choreiform  movements  have  been  observed  after  injection.  The 
organism  is  most  easily  obtained  from  the  substance  of  inflamed 
synovial  membrane  where  it  is  invading  the  tissues ;  a  part 
where  there  is  special  congestion  should  be  selected  as  being 
most  likely  to  give  positive  results.  It  is  only  occasionally  to 
be  obtained  from  the  fluid  in  joints.  It  has  also  been  cultivated 
from  the  blood  in  rheumatic  fever,  from  the  vegetations  on  the 
heart  valves,  and  from  other  acute  lesions ;  in  many  cases,  how- 
ever, cultures  from  the  blood  give  negative  results.  Beattie  in 
a  recent  paper  has  shown  that  in  rabbits  the  arthritis  produced 
reproduces  the  main  features  of  acute  or  subacute  rheumatism 
in  man,  namely,  the  rapidity  with  which  the  affection  flies  from 
joint  to  joint,  the  tendency  to  relapses,  the  contributory  effects 
of  exposure  to  cold,  and  the  absence  of  gross  anatomical  changes 
in  the  joints.  Poynton  and  Paine  cultivated  the  streptococcus 
from  the  cerebro-spinal  fluid  in  three  cases  where  chorea  was 
present,  and  also  detected  it  in  the  membranes  of  the  brain. 
They  consider  that  this  disease  is  probably  of  the  nature  of  a 
slight  meningo-myelitis  produced  by  the  organism.  The  facts 
already  accumulated  speak  strongly  in  favour  of  this  organism 
being  causally  related  to  rheumatic  fever,  though  this  cannot 
be  considered  completely  proved.  Andrewes  finds  that  the 
organism  has  the  same  cultural  characters  and  fermentative 
effects  as  the  streptococcus  fiecalis,  a  common  inhabitant  of  the 
intestine.  Even,  however,  if  the  two  organisms  were  the  same, 
it  might  well  be  j»ossible  that  rheumatic  fever  is  due  to  an 
infection  of  the  tissues  by  this  variety  of  streptococcus.  The 
clinical  data,  in  fact,  rather  point  to  rheumatic  fever  being 
due  to  an  infection  by  some  organism  frequently  present  in  the 


222          INFLAMMATION  AND  SUPPURATION 

body,  brought  about  by  some  state  of  predisposition  or  acquired 
susceptibility. 

Vaccination  Treatment  of  Infections  by  the  Pyogenic  Cocci. 
— From  his  study  of  the  part  played  by  phagocytosis  in  the 
successful  combat  of  the  pyogenic  bacteria  by  the  body,  Wright 
was  led  to  advocate  the  treatment  of  such  infections  during  their 
course  by  active  immunisation  by  means  of  dead  cultures  of  the 
infecting  agent  (for  methods  of  preparation  see  p.  133).  The 
treatment  is  applicable  when  the  infection  is  practically  local, 
as  in  acne  pustules,  in  boils,  etc.  (For  the  theoretical  questions 
raised,  see  Immunity.)  For  an  isolated  faruncle,  Wright  recom- 
mends a  dose  of  100  million  staphylococci  to  be  followed  three  or 
four  days  later  by  the  injection  of  250  to  300  millions,  and  for  an 
incipient  streptococcic  lymphangitis  a  dose  of  2,000,000  strepto- 
cocci. In  chronic  infections  the  number  of  bacteria  used  for  an 
injection  is  from  250,000,000  to  500,000,000,  but  a  smaller 
number  may  give  a  good  result,  and  the  general  principle  to  be 
adopted  is  to  use  the  smallest  dose  necessary  for  a  therapeutic 
effect.  If  repeated  injections  are  necessary,  Wright  recommends 
that  the  opsonic  index  should  be  observed  every  few  days  and 
the  injections  only  practised  during  a  positive  phase.  If  it  is 
not  practicable  to  use  the  strain  derived  from  the  lesion  for 
the  preparation  of  the  vaccine,  then  laboratory  cultures  or  the 
stock  vaccines  which  are  now  in  the  market  may  be  used ;  in 
such  cases  it  is  well  to  use  a  vaccine  made  from  a  mixture  of 
strains ;  in  skin  infections  a  mixture  of  staphylococcus  aureus 
and  albus  may  be  employed.  Such  means  have  been  extensively 
used  in  the  treatment  of  acne,  boils,  sycosis,  infections  of  the 
genito-urinary  tract  by  the  b.  coli,  infections  of  joints  by  the 
gonococcus,  and  in  many  cases  considerable  success  has  followed 
the  treatment.  Sometimes  acute  affections,  e.g.  puerperal 
septicaemia,  have  been  treated  by  vaccines.  It  is  difficult  here 
to  judge  of  the  results  which  have  been  attained,  but  in  any 
case  only  very  small  doses  of  the  vaccine  should  at  first,  at  any 
rate,  be  administered. 

Methods  of  Examination  in  Inflammatory  and  Suppurative 
Conditions. — These  are  usually  of  a  comparatively  simple  nature, 
and  include  (1)  microscopic  examination,  (2)  the  making  of 
cultures. 

(1)  The  pus  or  other  fluids  should  be  examined  microscopic- 
ally, first  of  all  by  means  of  film  preparations  in  order  to 
determine  the  characters  of  the  organisms  present.  The  films 
should  be  stained  (a)  by  one  of  the  ordinary  solutions,  such 
as  carbol-thionin-blue  (p.  105),  or  a  saturated  watery  solution  of 


METHODS  OF  EXAMINATION  223 

methylene-blue ;  and  (b)  by  Gram's  method.  The  use  of  the 
latter  is  of  course  of  high  importance  as  an  aid  in  the  recognition. 

(2)  As  most  of  the  pyogenic  organisms  grow  readily  on  the 
gelatin  media  at  ordinary  temperatures,  pure  cultures  can  be 
readily  obtained  by  the  ordinary  plate  methods.  But  in  many 
cases  the  separation  can  be  affected  much  more  rapidly  by  the 
method  of  successive  streaks  on  agar  tubes,  which  are  then 
incubated  at  37°  C.  When  the  presence  of  pneumococci  is 
suspected,  this  method  ought  always  to  be  used,  and  it  is  also  to 
l>e  preferred  in  the  case  of  streptococci.  Inoculation  experiments 
may  be  carried  out  as  occasion  arises. 

In  cases  of  suspected  blood  infection  the  examination  of  the 
blood  is  to  be  carried  out  by  the  methods  already  described 
(p.  72). 


CHAPTER  VIII. 

INFLAMMATORY  AND  SUPPURATIVE  CONDITIONS, 
CONTINUED:  THE  ACUTE  PNEUMONIAS,  EPI- 
DEMIC CEREBRO-SPINAL  MENINGITIS. 

Introductory. — The  term  Pneumonia  is  applied  to  several  con- 
ditions which  present  differences  in  pathological  anatomy  and  in 
origin.  All  of  these,  however,  must  be  looked  on  as  varieties  of 
inflammation  in  which  the  process  is  modified  in  different  ways, 
depending  on  the  special  structure  of  the  lung  or  of  the  parts 
which  compose  it.  There  is,  first  of  all,  and,  in  adults,  the  com- 
monest type,  the  acute  croupous  or  lobar  pneumonia,  in  which 
an  inflammatory  process  attended  by  abundant  fibrinous  exuda- 
tion affects,  by  continuity,  the  entire  tissue  of  a  lobe  or  of  a 
large  portion  of  the  lung.  It  departs  from  the  course  of  an 
ordinary  inflammation  in  that  the  reaction  of  the  connective 
tissue  of  the  lung  is  relatively  slight,  and  there  is  usually  no 
tendency  for  organisation  of  the  inflammatory  exudation  to 
take  place.  Secondly,  there  is  the  acute  catarrhal  or  lobular 
pneumonia,  where  a  catarrhal  inflammatory  process  spreads  from 
the  capillary  bronchi  to  the  air  vesicles,  and  in  these  a  change, 
consisting  largely  of  proliferation  of  the  endothelium  of  the 
alveoli,  takes  place  which  leads  to  consolidation  of  patches  of 
the  lung  tissue.  Up  till  1889  acute  catarrhal  pneumonia  was 
comparatively  rare  except  in  children.  In  adults  it  was  chiefly 
found  as  a  secondary  complication  to  some  condition  such  as 
diphtheria,  typhoid  fever,  etc.  Since,  however,  influenza  in  an 
epidemic  form  has  become  frequent,  catarrhal  pneumonia  has 
been  of  much  more  frequent  occurrence  in  adults,  has  assumed 
a  very  fatal  tendency,  and  has  presented  the  formerly  quite 
unusual  feature  of  being  sometimes  the  precursor  of  gangrene 
of  the  lung.  Besides  these  two  definite  types  other  forms  also 
occur.  Thus  instead  of  a  fibrinous  material  the  exudation  may 
be  of  a  serous,  hemorrhagic,  or  purulent  character.  Cases  of 
mixed  fibrinous  and  catarrhal  pneumonia  also  occur,  and 

224 


TYPES  OF  PNEUMONIA  225 

in  the  catarrhal  there  may  be  great  leucocytic  emigration. 
Hemorrhages  also  may  occur  here. 

Besides  the  two  chief  types  of  pneumonia  there  is  another 
group  of  cases  which  are  somewhat  loosely  denominated  septic 
pneumonias,  and  which  may  arise  in  two  ways :  (1)  by  the 
entrance  into  the  trachea  and  bronchi  of  discharges,  blood,  etc., 
which  form  a  nidus  for  the  growth  of  septic  organisms ;  these 
often  set  up  a  purulent  capillary  bronchitis  and  lead  to  infection 
of  the  air  cells  and  also  of  the  interstitial  tissue  of  the  lung ;  (2) 
from  secondary  pyogenic  infection  by  means  of  the  blood  stream 
from  suppurative  foci  in  other  parts  of  the  body.  (See  chapter 
on  Suppuration,  etc.)  In  these  septic  pneumonias  various 
changes,  resembling  those  found  in  the  other  types,  are  often 
seen  round  the  septic  foci. 

In  pneumonias,  therefore,  there  may  be  present  a  great  variety 
of  types  of  inflammatory  reaction.  We  shall  see  that  with  all  of 
them  bacteria  have  been  found  associated.  Special  importance 
is  attached  to  acute  croupous  pneumonia  on  account  of  its  course 
and  characters,  but  reference  will  also  be  made  to  the  other 
forms. 

Historical. — Acute  lobar  pneumonia  for  long  was  supposed  to  be  an 
effect  of  exposure  to  cold  ;  but  many  observers  were  dissatisfied  with 
this  view  of  its  etiology.  Not  only  did  cases  occur  where  no  such 
exposure  could  be  traced,  but  it  had  been  observed  that  the  disease 
sometimes  occurred  epidemically,  and  was  occasionally  contracted  by 
hospital  patients  lying  in  beds  adjacent  to  those  occupied  by  pneumonia 
oases.  Further,  the  sudden  onset  and  definite  course  of  the  disease  con- 
formed to  the  type  of  an  acute  infective  fever  ;  it  was  thus  suspected  by 
some  to  be  due  to  a  specific  infection.  This  view  of  its  etiology  was 
promulgated  in  1882-83  by  Friedliinder,  whose  results  were  briefly  as 
follows.  In  pneumonic  lungs  there  were  cocci,  adherent  usually  in 
pairs,  and  possessed  of  a  definitely  contoured  capsule.  These  cocci 
could  be  isolated  and  grown  on  gelatin,  and  on  inoculation  in  mice  they 
produced  a  kind  of  septicaemia  with  inflammation  of  the  serous  membranes. 
The  blood  and  the  exudation  in  serous  cavities  contained  numerous 
capsulated  diplococci.  There  is  little  doubt  that  many  of  the  organisms 
seen  by  Friedlander  were  really  Fraenkel's  pneumococcus,  to  be  presently 
described. 

By  many  observers  it  had  been  found  that  the  sputum  of  healthy 
men,  when  injected  into  animals,  sometimes  caused  death,  with  the 
same  symptoms  as  in  the  case  of  the  injection  of  Friedliinder's  coccus  ; 
and  in  the  blood  and  serous  exudations  of  such  animals  capsulated 
diplococci  were  found.  A.  Fraenkel  found  that  the  sputum  of  pneumonic 
patients  was  much  more  fatal  and  more  constant  in  its  effects  than  that 
of  healthy  individuals.  The  cocci  which  were  found  in  animals  dead  of 
this  "sputum  septicaemia,"  as  it  was  called,  differed  from  Friedlander's 
cocci  in  several  respects,  to  be  presently  studied.  Fraenkel  further 
investigated  a  few  cases  of  pneumonia,  and  isolated  from  them  cocci 
identical  in  microscopic  appearances,  cultures,  and  pathogenic  effects, 


226  THE  ACUTE  PNEUMONIAS 

with  those  isolated  in  sputum  septicaemia.  The  most  extensive  investi- 
gations on  the  whole  question  were  those  of  Weichselbaum,  published 
in  1886.  This  author  examined  129  cases  of  the  disease,  including 
cases  not  only  of  acute  croupous  pneumonia,  but  of  lobular  and  septic 
pneumonia.  From  them  he  isolated  four  groups  of  organisms.  (1) 
Diplococcus  pneumonia;.  This  he  described  as  an  oval  or  lancet-formed 
coccus,  corresponding  in  appearance  and  growth  characters  to  Fraenkel's 
coccus.  (2)  Streptococcus  pneumonia?.  This  on  the  whole  presented 
similar  characters  to  the  last,  but  it  was  more  vigorous  in  its  growth, 
and  could  grow  below  20°  C. ,  though  it  preferred  a  temperature  of  37°  C. 
(3)  Staphylococcus  pyogenes  aureus.  (4)  Bacillus  pneumonice.  This  was 
a  rod-shaped  organism,  and  was  identical  with  Friedlander's  pneumo- 
coccus.  Of  these  organisms  the  diplococcus  pneumoniae  was  by  far  the 
most  frequent.  It  also  occurred  in  all  forms  of  pneumonia.  Next  in 
frequency  was  the  streptococcus  pneumonias,  and  lastly  the  bacillus 
pneumonias.  Inoculation  experiments  were  also  performed  by  Weichsel- 
baum with  each  of  the  three  characteristic  cocci  he  isolated.  The 
diplococcus  pneumonias  and  the  streptococcus  pneumonias  both  gave 
pathogenic  effects  of  a  similar  kind  in  certain  animals. 

The  general  result  of  these  earlier  observations  was  to  establish 
the  occurrence  in  connection  with  pneumonia  of  two  species  of 
organisms,  each  having  its  distinctive  characters,  viz.  : — 

1.  Fraenkel's  pneumococcus^  which  is  recognised  to  be  identical 
with  the  coccus  of  "  sputum  septicremia,"  with  Weichselbaum's 
diplococcus  pneumonias,  and  with  his  streptococcus  pneumonias. 

2.  Friedlander's  pneumococcus  (now  known  as  Friedlander's 
pneumobacillus),  which  is  almost  certainly  the  bacillus  pneu- 
moniae of  Weichselbaum. 

We  shall  use  the  terms  "  Fraenkel's  pneumococcus "  and 
"Friedlander's  pneumobacillus,"  as  these  are  now  usually 
applied  to  the  two  organisms. 

Microscopic  Characters  of  the  Bacteria  of  Pneumonia.— 
Methods. — The  organisms  present  in  acute  pneumonia  can  best 
be  examined  in  film  preparations  made  from  pneumonic  lung 
(preferably  from  a  part  in  a  stage  of  acute  congestion  or  early 
hepatisation),  or  from  the  gelatinous  parts  of  pneumonic  sputum 
(here  again  preferably  when  such  sputum  is  either  rusty  or 
occurs  early  in  the  disease),  or  in  sections  of  pneumonic  lung. 
Such  preparations  may  be  stained  by  any  of  the  ordinary  weak 
stains,  such  as  a  watery  solution  of  methylene-blue,  but  Gram's 
method  is  to  be  preferred,  with  Bismarck-brown  or  Ziehl-Neelsen 
carbol-f  uchsin  (one  part  to  ten  of  water)  as  a  contrast  stain ; 
with  the  latter  it  is  best  either  to  stain  for  only  a  few  seconds, 
or  to  overstain  and  then  decolorise  with  alcohol  till  the  ground 
of  the  preparation  is  just  tinted.  The  capsules  can  also  be 
stained  by  the  methods  already  described  (p.  109).  In  such 
preparations  as  the  above,  and  even  in  specimens  taken  from 


BACTERIA  IN  PNEUMONIA  227 

the  lungs  immediately  after  death  (as  may  be  quite  well  done 
by  means  of  a  hypodermic  syringe),  putrefactive  and  other 
bacteria  may  be  present,  but  those  to  be  looked  for  are  capsulated 
organisms,  which  may  be  of  either  or  both  of  the  varieties 
mentioned. 

(1)  FraenkeVs  Pneumococcus. — This  organism  occurs   in  the 
form  of   a   small  oval   coccus,  about    1  p.  in    longest  diameter, 
arranged  generally  in   pairs  (diplococci),  but  also  in  chains  of 
four  to  ten  (Fig.  62).     The  free  ends  are  often  pointed  like  a 
lancet,    hence    the    term 

diplococcus        lanceolatus  .%  ... 

has  also  been  applied  to  .X        '    ,  '/;    * 

it.     These  cocci,  in  their  •.•>"• 

typical  form,  have  round       /^  *  *  ''*    '. 

them  a  capsule,  which,  in     /j*  f     t         .  - 

films  stained  by  ordinary  ^f 

methods,  usually  appears    I  ,  „.«  ,  tf 

as  an  unstained  halo,  but    1  ;»>/ 

is  sometimes  stained  more    \  ji          v ,    t"        s,  s    ' 

deeply   than   the   ground     V?»;  ,*      * 

of  the  preparation.     This 

difference  in  staining  de-  >•/ 

pends,  in  part  at  least,  on 

the  amount  of  decolorisa- 

tiou  to  which  the  prepara-     m  ^  _p.lm  prepftrations  of  pneumonic 

tlOll    lias    been    subjected.          sputum,  showing  numerous  pneumococci 

The      capsule      is     rather         (Fraenkel's)    with     unstained    capsules ; 

Kr^urlor  tkan  tlia  KrtrJv  sonie  are  arranged  in  short  chains.  See 
broader  tnan  tne  boay  algo  plate  j  ^  F?g  2 

of  the  coccus,  and  has  a  Stained  with  carbol  fuchsin.     x  1000. 

sharply   defined    external 

margin.  Often  in  sputum  and  even  in  the  lung  no  capsule  can 
be  demonstrated.  The  organism  takes  up  the  basic  aniline 
stains  with  great  readiness,  and  also  retains  the  stain  in  Gram's 
method.  It  is  the  organism  of  by  far  the  most  frequent  occur- 
rence in  true  croupous  pneumonia,  and  in  fact  may  be  said  to 
be  rarely  absent. 

(2)  Friedldnder's  Pneumobacillus. — As  seen  in  the  sputum 
and  tissues,  this  organism,  both  in  its  appearance  and  arrange- 
ment, as  also  in  the  presence  of  a  capsule,  somewhat  resembles 
Fraenkel's  pneumococcus,   and  it  was  at  first  described  as  the 
"  pneumococcus."     The  form,  however,  is  more  of  a  short  rod- 
shape,  and  it  has  blunt  rounded  ends ;  it  is  also  rather  broader 
than  Fraenkel's  pneumococcus.     It  is  now  classed  amongst  the 

illi,  especially  in  view  of  the  fact  that  elongated  rod  forms 


228 


THE  ACUTE  PNEUMONIAS 


may    occur 


(Fig- 


FIG.  63.— Friedlander's  pneumobacillus,  show- 
ing the  variations  in  length,  also  capsules. 
Film  preparation  from  exudate  in  a  case  of 


pneumona, 


1000. 


pneumonia  than  Fraenkel's 
latter;    very    rarely    it 
occurs  alone. 

In  sputum  prepara- 
tions the  capsule  of 
both  pneurnobacteria 
may  not  be  recognis- 
able, and  the  same  is 
sometimes  true  of  lung 
preparations.  This  is 
probably  due  to  changes 
which  occur  in  the  cap- 
sule as  the  result  of 
changes  in  the  vitality 
of  the-  organisms.  Some- 
times in  preparations 
stained  by  ordinary 
methods  the  difficulty 
of  recognising  the  cap- 
sule when  it  is  present, 
is  due  to  the  refractive 
index  of  the  fluid  in 
which  the  specimen  is 


capsule  has  the  same  general 
characters  as  that  of 
Fraenkel's  organism. 
Friedlander's  pneumo- 
bacillus stains  readily 
with  the  basic  aniline 
stains,  but  loses  the 
stain  in  Gram's  method, 
and  is  accordingly  col- 
oured with  the  contrast 
stain, — fuchsin  or  Bis- 
marck-brown, as  above 
recommended.  A  valu- 
able means  is  thus 
afforded  of  distinguish- 
ing it  from  Fraenkel's 
pneumococcus  in  micro- 
scopic preparations. 

Friedlander's     organ- 
ism   is    much    less   fre- 
quently      present       in 
sometimes  it  is  associated  with  the 


FIG.  64. — Fraenkel's  pneumococcus  in  serous 
exudation  at  site  of  inoculation  in  a  rabbit, 
showing  capsules  stained. 
Stained  bv  Rd.  Muir's  method,      x  1000. 


mounted  being   almost  identical  with 


CULTIVATION  OF  PNEUMOCOCCTJS  229 

that  of  the  capsule.  This  difficulty  can  always  be  overcome  by 
having  the  groundwork  of  the  preparation  tinted. 

The  Cultivation  of  Fraenkel's  Pneumococcus. — It  is  usually 
difficult,  and  sometimes  impossible,  to  isolate  this  coccus  directly 
from  pneumonic  sputum.  On  culture  media  it  has  not  a  vigorous 
growth,  and  when  mixed  with  other  bacteria  it  is  apt  to  be 
overgrown  by  the  latter.  To  get  a  pure  culture  it  is  best  to 
insert  a  small  piece  of  the  sputum  beneath  the  skin  of  a  rabbit 
or  a  mm  isc.  In  about  twenty-four  to  forty -eight  hours  the 
animal  will  die,  with  numerous  capsul- 
ated  pneomooocci  throughout  its  blood. 
K PI  111  the  heart-blood  cultures  can  be 
easily  obtained.  Cultures  can  also  be 
got  post  mortem  from  the  lungs  of 
pneumonic  patients  by  streaking  a 
number  of  agar  or  blood-agar  tubes  with 
a  scraping  taken  from  the  area  of  acute 
congestion  or  commencing  red  hepatisa 
tion,  and  incubating  them  at  37°  ('. 
The  colonies  of  the  pneumococcus  appear 
as  almost  transparent  small  discs  which 
have  been  compared  to  drops  of  dew 
(Fig.  65).  This  method  is  also  some- 
times successful  in  the  case  of  sputum. 

The  appearances  presented  in  cultures  Fro.  K._Stroke  culture  of 
by  different  varieties  nt  the  pneuino-  Fraenkel's  pneumococcus 
coccus  vary  somewhat.  It  always  grows  °n  Mow  I  agar.  The 
best  „„  l,l,,o,l  son,,,,  ,,  „„  l>,ViHer'S  !*&££?**£ 
blood  agar.  It  usually  grows  well  on  four  hours'  growth  at 
ordinary  agar  or  in  bouillon,  but  not  :;7  r-  Natural  size. 
BO  well  on  glycerin  agar.  In  a  stroke 

culture  on  hlood  serum,  growth  appears  as  an  almost  trans- 
parent pellicle  along  the  track,  with  isolated  colonies  at  the 
margin.  On  agar  media  it  is  more  manifest,  but  otherwise 
has  similar  characters.  On  agar  plates  colonies  are  very 
transparent,  but  under  a  low  power  of  the  microscojK3  appear 
to  have  a  compact  finely  granular  centre  and  a  pale  trans- 
parent periphery.  The  appearances  arc  similar  to  those  of 
a  culture  of  streptococcus  pyogenes,  but  the  growth  is  less 
vigorous,  and  is  more  delicate  in  appearance.  A  similar 
statement  also  applies  to  cultures  in  f/elatin  at  2'2°  C.,  growth 
in  a  stab  culture  appearing  as  a  row  of  minute  points  which 
remain  of  small  size;  there  is,  of  course,  no.  liquefaction  of  the 
medium.  In  bouillon,  growth  forms  a  slight  turbidity,  \\hich 


230  THE  ACUTE  PNEUMONIAS 

settles  to  the  bottom  of  the  vessel  as  a  slight  dust-like  deposit. 
On  potatoes,  as  a  rule,  no  growth  appears.  Cultures  may  be 
maintained  for  one  or  two  months,  if  fresh  sub-cultures  are  made 
every  four  or  five  days,  but  they  tend  ultimately  to  die  out. 
They  also  rapidly  lose  their  virulence,  so  that  four  or  five  days 
after  isolation  from  an  animal's  body  their  pathogenic  action 
is  already  diminished.  Eyre  and  Washbourn,  however,  have 
succeeded  in  maintaining  cultures  in  a  condition  of  constant 
virulence  for  at  least  three  months  by  growing  the  organisms 

on    agar    smeared    with 

\  rabbit  blood.     The  agar 

I     *  must  be  prepared  with 

A'     £**  *s    N  Witte's    peptone,     must 

/  V   v  not  be  heated  over  100° 

^  ^     /  C.,  and  after  neutralisa- 

|       s  -*  «^«*        V»  tion   (rosolic  acid  being 

i        k  t  ;$y     used    as    the    indicator) 

must  have  '5  per  cent,  of 

^  **    \  normal  sodium  hydrate 

N  V^  added.     The  tubes  when 

\  v»  %  inoculated  are  to  be  kept 

r    *.  \  at   37°*5  C.  and   sealed 

i^  %  to  prevent  evaporation. 

In      ordinary     artificial 

•*  '  media  pneumococci  usu- 

FIG.  66. — Fraenkel's  pueumococcus  from  a  pure    ally  appear  as  diplococci 
culture  on  blood  agar  of  twenty-four  hours'    w:tilont    fl    Par>milp     but 
growth,  some  in  pairs,  some  in  short  chains.      Wltnout    a    capsule,    t 
Stained  with  weak  carbol-fuchsin.     x  1000.       in     preparations     made 

from  the  surface  of  agar 

or  from  bouillon,  shorter  or  longer  chains  may  be  observed 
(Fig.  66).  After  a  few  days'  growth  they  lose  their  regular  shape 
and  size,  and  involution  forms  appear.  Usually  the  pneumo- 
coccus  does  not  grow  below  22°  C.,  but  forms  in  which  the 
virulence  has  disappeared  often  grow  well  at  20°  C.  Its  optimum 
temperature  is  37°  C.,  its  maximum  42°  C.  It  is  preferably  an 
aerobe,  but  can  exist  without  oxygen.  It  prefers  a  slightly  alka- 
line medium  to  a  neutral,  and  does  not  grow  on  an  acid  medium. 
In  ordinary  media  the  pneumococcus  does  not  usually  appear  to 
develop  a  capsule,  but  according  to  Hiss,  the  absence  of  a  capsule 
is  often  only  apparent,  and  if  in  making  cover-glass  preparations 
off  such  media  some  ox  or  rabbit  serum  be  used  as  the  diluent, 
and  the  films  stained  by  his  copper-sulphate  method  (p.  109),  a 
capsule  can  be  demonstrated.  Capsulation  frequently  appears 
in  fluid  serum  media,  e.g.,  can  be  readily  recognised  if  the 


CULTIVATION  OF  PNEUMOCOCCUS  231 

organism  be  grown  in  rabbit  or  human  serum  which  has  been 
obtained  under  aseptic  precautions  and  heated  for  half  an  hour 
at  55°  C. 

The  pneuinococcus  ferments  saccharose,  raffinose,  and  lactose, 
and  a  similar  fermentative  action  on  inuliri  is  important,  as 
ordinary  streptococci  do  not  so  readily  ferment  this  sugar. 
Apparently  some  samples  of  inulin  are  more  readily  acted  on 
than  others.  Usually  the  test  is  carried  out  with  Hiss's  inulin 
serum  water  medium,  in  which  coagulation  of  the  serum  results 
(p.  47),  but  some  investigators  have  had  more  success  with 
inulin  bouillon,  acid  production  being  estimated  by  titration 
against  soda  with  a  phenolphthalein  indicator. 

There  has  been  described  by  Eyre  and  Washbourn  a  non- 
pathogenic  type  of  the  pneumococcus  which  may  be  found  in 
the  healthy  mouth,  and  which  may  also  be  produced  during  the 
saprophytic  growth  of  the  virulent  form.  From  the  latter  it 
differs  generally  in  its  more  vigorous  growth,  in  producing  a 
uniform  cloud  in  bouillon,  in  slowly  liquefying  gelatin,  and  in 
growing  on  potato.  The  facts  that  in  cultures  the  pneumococcus 
often  grows  in  chains,  and  that  occasionally  streptococci  are 
found  to  develop  capsules,  have  raised  the  question  of  the  rela- 
tionship of  the  pneumococcus  to  other  streptococci.  When, 
however,  biological  characters  are  taken  along  with  morpho- 
logical, relatively  little  difficulty  arises  in  the  recognition  of  a 
true  pneumococcus.  Here  the  reaction  in  inulin  is  important. 
It  may  be  said  that  the  capacity  of  a  capsulated  organism  to 
produce  acid  from  this  sugar  makes  its  being  a  true  pneumococcus 
extremely  probable.  That  the  pneumococcus  may  be  related  to 
other  streptococci  is,  however,  shown  by  the  fact  that  both  sets 
of  organisms  tend  to  originate  common  group  agglutinins. 

Considerable  attention  has  been  devoted  to  a  bacterium 
originally  described  by  Schottmiiller,  and  called  by  him  the 
Streptococcus  mucosus.  This  organism  has  been  isolated  from 
a  variety  of  suppurative  conditions  and  also  from  certain  cases  of 
pneumonia.  In  culture,  it  diners  from  the  pneumococcus  in  the 
colonies  being  more  clear,  transparent,  and  dewdrop-like,  showing 
great  tendency  to  confluence,  and  being  more  slimy  than  those 
of  the  pneumococcus.  It  coagulates  the  serum  in  Hiss's  inulin 
serum  water  medium.  It  is,  pathogenic  to  white  mice,  but  its 
pathogenicity  in  the  rabbit  seems  to  be  less  than  that  of  the  true 
pneumococcus.  Its  agglutinative  reactions  are  somewhat  pecu- 
liar. Unlike  the  pneumococcus,  it  produces  in  animals  only  a 
weak  agglutinating  serum,  but  such  a  serum  often  can  agglu- 
tinate pneumococci.  Further,  antipneumococcal  sera  frequently 


232  THE  ACUTE  PNEUMONIAS 

agglutinate  the  streptococcus  mucosus  more  readily  than  other 

streptococci.     All  the  facts  seem  to  point  to  this  organism  being 

closely  allied  to  the  true  pneumococcus. 

The   Cultivation    of  Friedlander's   Pneumobacillus. — This 

organism,  when  present  in  sputum  or  in  a  pneumonic  lung,  can 
...     i~<  be  readily  separated  by  making  ordinary 

gelatin  plate  cultures,  or  a  series  of  suc- 
cessive strokes  on  agar  tubes.  The  surface 
colonies  always  appear  as  white  discs 
which  become  raised  from  the  surface  so 
as  to  resemble  little  knobs  of  ivory. 
From  these,  pure  cultures  can  be  readily 
obtained.  The  appearance  of  a  stab  cul- 
ture in  gelatin  is  very  characteristic. 
At  the  site  of  the  puncture,  there  is  on 
the  surface  a  white  growth  heaped  up, 
it  may  be  fully  one-eighth  of  an  inch, 
_,;.;,%  above  the  level  of  the  gelatin;  along  the 

needle  track  there  is  a  white  granular 
appearance,  so  that  the  whole  resembles 
a  white  round-headed  nail  driven  into 
the  gelatin  (Fig.  67).  Hence  the  name 
"  nail-like "  which  has  been  applied. 
Occasionally  bubbles  of  gas  develop  along 
the  line  of  growth.  There  is  no  lique- 
faction of  the  medium.  On  sloped  agar 
it  forms  a  very  white  growth  with  a 
shiny  lustre,  which,  when  touched  with 
a  platinum  needle,  is  found  to  be  of  a 
viscous  consistence.  In  cultures  much 
^>i  i  M*^  longer  rods  are  formed  than  in  the 

FIG.  67.-Stab  culture  of  tisSUGS  °f  the  body  (FiS'  68)'  On  tlle 
Friedlander's  pneumo-  surface  of  potatoes  it  forms  an  abundant 
bacillus  in  peptone  moist  white  layer.  It  is  non-motile. 
nalSe  "appearance  •  Friedlander's  bacillus  has  active  ferment- 
ten  days'  growth.'  ing  powers  on  sugars,  though  varieties 
Natural  size.  isolated  by  different  observers  vary  in 

the  degree  in  which  such  powers  are 
possessed.  It  always  seems  capable  of  acting  on  dextrose, 
lactose,  maltose,  dextrin,  and  mannite,  and  sometimes  also  on 
glycerin.  The  substances  produced  by  the  fermentation  vary 
with  the  sugar  fermented,  but  include  ethylic  alcohol,  acetic 
acid,  laevolactic  acid,  succinic  acid,  along  with  hydrogen  and 
carbonic  acid  gas.  The  amount  of  acid  produced  from  lactose 


OCCURRENCE  OF  PNEUMOBACTERTA         233 

seems  only  exceptionally  sufficient  to  cause  coagulation  of  milk. 
With  regard  to  indol  formation  the  results  of  different  observers 
vary.     Here,  as  with  other  reactions,  it  is  to  be  noted  that  only 
strains      isolated      from 
cases   <>t    pneumonia  are 
to  be  taken  into  account. 
It  is  said  by  some  that 
the  bacillus  is  identical 
with    an   organism   com- 
mon  in  sour  milk,   and 
also  a  normal  inhabitant 
of   the  human  intestiiu', 
namely,     the    bacterium 


The  Occurrence  of 
the  Pneumobacteria  in 
Pneumonia  and  other 
Conditions.  Capsulated 
organisms  have  been  FlG-  68.— Friedliinder's  pueumobacillus,i 
c  i  >  •  from  a  young  culture  on  agar,  showing 

found    m   every   variety          some  n.d -shaped  forms. 

of   the  disease — in   acute          Stained  \vith  thionin-blue.      x  1000. 

croupous   pneumonia,  in 

broncho-pneumonia,  in  septic  pneumonia.  In  the  great  majority 
of  these  it  is  Fraenkel's  pneumococcus  which  both  microscopic- 
ally and  culturally  has  been  found  to  be  present.  Fried  lander's 
pneumobacillus  occurs  in  only  about  5  per  cent,  of  the  cases. 
It  may  be  present  alone  or  associated  with  Fraenkel's  organism. 
In  a  case  of  croupous  pneumonia  the  pneumococci  are  found  all 
through  the  affected  area  in  the  lung,  especially  in  the  exudation 
in  the  air-cells.  They  also  occur  in  the  pleural  exudation  and 
effusion,  and  in  the  lymphatics  of  the  lung.  The  greatest  number 
are  found  in  the  parts  where  the  inflammatory  process  is  most 
recent,  e.g.  in  an  area  of  acute  congestion  in  a  case  of  croupous 
pneumonia,  and  therefore  such  parts  are  preferably  to  be  selected 
for  microscopic  examination,  and  as  the  source  of  cultures. 
Sometimes  there  occur  in  pneumonic  consolidation  areas  of 
suppurative  softening,  which  may  spread  diffusely.  In  such 
areas  the  pneuniococci  occur  with  or  without  ordinary  pyogenic 
organisms,  streptococci  being  the  commonest  concomitants.  In 

1  The  apparent  size  of  this  organism,  on  account  of  the  nature  of  its  sheath. 
varies  much  according  to  the  stain  used.  1 1'  stained  with  a  strong  stain,  e.g. 
carhol-fuchsin,  its  thi<-kne^  appears  nearly  twice  as  great  as  is  shown  in  the 
tigure. 


234  THE  ACUTE  PNEUMONIAS 

other  cases,  especially  when  the  condition  is  secondary  to  in- 
fluenza, gangrene  may  supervene  and  lead  to  destruction  of  large 
portions  of  the  lung.  In  these  a  great  variety  of  bacteria,  both 
aerobes  and  anaerobes  are  to  be  found. 

In  ordinary  broncho-pneumonias  also  Fraenkel's  pneumo- 
coccus  is  usually  present,  sometimes  along  with  pyogenic  cocci ; 
in  the  broncho-pneumonias  secondary  to  diphtheria  it  may  be 
accompanied  by  the  diphtheria  bacillus,  and  also  by  pyogenic 
cocci ;  in  typhoid  pneumonias  the  typhoid  bacillus  or  the  b.  coli 
may  be  alone  present  or  be  accompanied  by  the  pneurno- 
coccus,  and  in  influenza  pneumonias  the  influenza  bacillus  may 
occur.  In  septic  pneumonias  the  pyogenic  cocci  in  many  cases 
are  the  only  organisms  discoverable,  but  the  pneumococcus  may 
also  be  present.  Especially  important,  as  we  shall  see,  from  the 
point  of  view  of  the  etiology  of  the  disease,  is  the  occurrence  in 
other  parts  of  the  body  of  pathological  conditions  associated 
with  the  presence  of  the  pneumococcus.  By  direct  extension  to 
neighbouring  parts  empyema,  pericarditis,  and  lymphatic  enlarge- 
ments in  the  mediastinum  and  neck  may  take  place ;  in  the  first 
the  pneumococcus  may  occur  either  alone  or  with  pyogenic  cocci. 
But  distant  parts  may  be  affected,  and  the  pneumococcus  may 
be  found  in  suppurations  and  .inflammations  in  various  parts  of 
the  body  (subcutaneous  tissue,  peritoneum  (especially  in  children), 
joints,  kidneys,  liver,  etc.),  in  otitis  media,  ulcerative  endocarditis 
(p.  216),  and  meningitis.  In  fact,  there  is  practically  no  inflam- 
matory or  suppurative  condition  in  the  body  in  which  the 
pneumococcus  in  pure  culture  may  not  be  found.  These  condi- 
tions may  take  place  either  as  complications  of  pneumonia,  or 
they  may  constitute  the  primary  disease.  The  occurrence  of 
meningitis  is  of  special  importance,  for  next  to  the  lungs  the 
meninges  appear  to  be  the  parts  most  liable  to  attack  by  the 
pneumococcus.  A  large  number  of  cases  have  been  investigated 
by  Netter,  who  gives  the  following  tables  of  the  relative  fre- 
quency of  the  primary  infections  by  the  pneumococcus  in  man  : — 

(1)  In  adults- 
Pneumonia        .         .         .         .         .         .         .     65 '95  per  cent. 

Broncho-pneumonia)  15 -85 

Capillary  bronchitis) 

Meningitis .  •  13'00 

Empyema         .         .         .         .         .         .         .       8'53 

Otitis 2-44 

Endocarditis 1'22 

Liver  abscess     .         .         .         .         .         .         ,1'22 

(2)  In  children  46  cases  were  investigated.     In  29  the  primary  affection 
was  otitis  media,  in  12  broncho-pneumonia,  in  2  meningitis,  in  1  pneu- 
monia, in  1  pleurisy   in  1  pericarditis. 


EXPERIMENTAL  INOCULATION  235 

Thus  in  children  the  primary  source  of  infection  is  in  a  great 
many  cases  an  otitis  media,  and  Netter  concludes  that  infection 
takes  place  in  such  conditions  from  the  nasal  cavities. 

As  bearing  on  the  occurrence  of  pneumococcal  infections 
.secondary  to  such  a  local  lesion  as  pneumonia,  it  is  important  to 
note  that  in  a  large  proportion  of  cases  of  the  latter  disease  the 
jineumococcus  can  be  isolated  from  the  blood. 

Experimental  Inoculation. — The  pneumococcus  of  Fraenkel  is 
pathogenic  to  various  animals,  though  the  effects  vary  somewhat 
\vith  the  virulence  of  the  race  used.  The  susceptibility  of 
different  species,  as  Gamaleia  has  shown,  varies  to  a  considerable 
extent.  The  rabbit,  and  especially  the  mouse,  are  very  sus- 
ceptible ;  the  guinea-pig,  the  rat,  the  dog,  and  the  sheep 
occupy  an  intermediate  position ;  the  pigeon  is  immune.  In 
the  more  susceptible  animals  the  general  type  of  the  disease 
produced  is  not  pneumonia,  but  a  general  septiccemia.  Thus,  if 
a  rabbit  or  a  mouse  be  injected  subcutaneously  with  pneumonic 
sputum,  or  with  a  scraping  from  a  pneumonic  lung,  death 
occurs  in  from  twenty-four  to  forty-eight  hours.  There  is  some 
tibrinous  infiltration  at  the  point  of  inoculation,  the  spleen  is 
often  enlarged  and  firm,  and  the  blood  contains  capsulated 
pneumococci  in  large  numbers  (Fig.  69).  If  the  seat  'of  inocula- 
tion be  in  the  lung,  there  generally  results  pleuritic  effusion  on 
both  sides,  and  in  the  lung  there  may  be  a  process  somewhat 
resembling  the  early  stage  of  acute  croupous  pneumonia  in  man. 
There  are  often  also  pericarditis  and  enlargement  of  spleen. 
We  have  already  stated  that  cultures  of  the  pneumococcus  on 
artificial  media  in  a  few  days  begin  to  lose  their  virulence. 
Now,  if  such  a  partly  attenuated  culture  be  injected  sub- 
cutaneously into  a  rabbit,  there  is  greater  local  reaction ; 
pneumonia,  with  exudation  of  lymph  on  the  surface  of  the 
pit-lira,  and  a  similar  condition  in  the  peritoneum,  may  occur. 
In  sheep  greater  immunity  is  marked  by  the  occurrence,  after 
subcutaneous  inoculation,  of  an  enormous  local  sero-fibrinous 
exudation,  and  by  the  fact  that  few  pneumococci  are  found  in 
the  blood  stream.  Intra-pulmonary  injection  in  sheep  is 
followed  by  a  typical  pneumonia,  which  is  generally  fatal.  The 
dog  is  still  more  immune ;  in  it  also  intra-pulmonary  injection  is 
followed  by  a  fibrinous  pneumonia,  which  is  only  sometimes 
fatal.  Inoculation  by  inhalation  appears  only  to  have  been 
performed  in  the  susceptible  mouse  and  rabbit;  here  also 
septicaemia  resulted. 

The  general  conclusion  to  be  drawn  from  these  experiments 
thus  is  that  in  highly  susceptible  animals  virulent  pneumococci 


236 


THE  ACUTE  PNEUMONIAS 


produce  a  general  septicaemia  ;  whereas  in  more  immune  species 
there  is  an  acute  local  reaction  at  the  point  of  inoculation,  and 
if  the  latter  be  in  the  lung,  then  there  may  result  pneumonia, 
which,  of  course,  is  merely  a  local  acute  inflammation  occurring 
in  a  special  tissue,  but  identical  in  essential  pathology  with  an 
inflammatory  reaction  in  any  other  part  of  the  body.  When  a 
dose  of  pneumococci  sufficient  to  kill  a  rabbit  is  injected  sub- 


FIG.  69. — Capsulated  pueumococcus  in  blood  taken  from  the  heart 
of  a  rabbit,  dead  after  inoculation  with  pneumonic  sputum. 

Dried  film,  fixed  with  corrosive  sublimate.  Stained  with  oarbol- 
fuchsin  and  partly  decolorised,  x  1000. 

cutaneously  in  the  human  subject,  it  gives  rise  to  a  local  inflam- 
matory swelling  with  redness  and  slight  rise  of  temperature,  all 
of  which  pass  off  in  a  few  days.  It  is  therefore  justifiable  to 
suppose  that  man  occupies  an  intermediate  place  in  the  scale  of 
susceptibility,  probably  between  the  dog  and  the  sheep,  and 
that  when  the  pneumococcus  gains  an  entrance  to  his  lungs  the 
local  reaction  in  the  form  of  pneumonia  occurs.  In  this  con- 
nection the  occurrence  of  manifestations  of  general  infection 
associated  with  pneumonia  in  man  is  of  the  highest  import- 


EXPERIMENTAL  INOCULATION  237 

ance.  We  have  seen  that  meningitis  and  other  inflammations 
are  not  very  rare  complications  of  the  disease,  and  such  cases 
form  a  link  connecting  the  local  disease  in  the  human  subject 
with  the  general  septicaemic  processes  which  may  be  produced 
artificially  in  the  more  susceptible  representatives  of  the  lower 
animals. 

A  fact  which  at  first  appeared  rather  to  militate  against  the 
pneumococcus  being  the  cause  of  pneumonia  was  the  discovery 
of  this  organism  in  the  saliva  of  healthy  men.  This  fact  was 
early  pointed  out  by  Pasteur,  and  also  by  Fraenkel,  and  the 
observation  has  been  confirmed  by  many  other  observers.  It 
can  certainly  be  isolated  from  the  mouths  of  a  considerable 
proportion  of  normal  men,  from  their  nasal  cavities,  etc.,  being 
probably  in  any  particular  individual  more  numerous  at  some 
times  (especially,  it  is  stated,  during  the  winter  months,  i.e.  a 
little  before  the  period  of  the  greatest  prevalence  of  pneumonia) 
than  at  others,  HI  id  sometimes  being  entirely  absent.  This 
can  be  proved,  of  course,  by  inoculation  of  susceptible  animals. 
Such  a  fact,  however,  only  indicates  the  importance  of  pre- 
disposing causes  in  the  etiology  of  the  disease,  and  it  is  further 
to  be  observed  that  we  have  corresponding  facts  in  the  case  of 
the  diseases  caused  by  pyogenic  staphylococci,  streptococci,  the 
bacillus  coli,  etc.  It  is  probable  that  by  various  causes  the 
vitality  and  power  of  resistance  of  the  lung  are  diminished,  and 
that  then  the  pneumococcus  gains  an  entrance.  In  relation  to 
this  possibility  we  have  the  very  striking  fact  that  in  the 
irregular  forms  of  pneumonia,  secondary  to  such  conditions  as 
typhoid  and  diphtheria,  the  pneumococcus  is  very  frequently 
present,  alone  or  with  other  organisms.  Apparently  the  effects 
produce;!  by  such  bacteria  as  the  b.  typhosus  and  the  b. 
diphtherias  can  devitalise  the  lung  to  such  an  extent  that 
secondary  infection  by  the  pneumococcus  is  more  likely  to  occur 
and  set  up  pneumonia.  We  can  therefore  understand  how 
much  less  definite  devitalising  agents  such  as  cold,  alcoholic 
excess,  etc.,  can  play  an  important  part  in  the  causation  of 
pneumonia.  In  this  way  also  other  abnormal  conditions  of  the 
respiratory  tract,  a  slight  bronchitis,  etc.,  may  play  a  similar 
part. 

It  is  more  difficult  to  explain  why  sometimes  the  pneumo- 
coccus is  associated  with  a  spreading  inflammation,  as  in  croupous 
pneumonia,  whilst  at  other  times  it  is  localised  to  the  catarrhal 
patches  in  broncho-pneumonia.  It  is  quite  likely  that  in  the 
former  condition  the  organism  is  possessed  of  a  different  order 
of  virulence,  though  of  this  \ve  have  no  direct  proof.  We  have, 


238  THE  ACUTE  PNEUMONIAS 

however,  a  closely  analogous  fact  in  the  case  of  erysipelas ;  this 
disease,  we  have  stated  reasons  for  believing,  is  produced  by 
a  streptococcus  which,  when  less  virulent,  causes  only  local 
inflammatory  and  suppurative  conditions. 

Summary. — We  may  accordingly  summarise  the  facts  re- 
garding the  relation  of  Fraenkel's  pneumococcus  to  the  disease 
by  saying  that  it  can  be  isolated  from  nearly  all  cases  of  acute 
croupous  pneumonia,  and  also  from  a  considerable  proportion 
of  other  forms  of  pneumonia.  When  injected  into  the  lungs  of 
moderately  insusceptible  animals  it  gives  rise  to  pneumonia.  If, 
in  default  of  the  crucial  experiment  of  intra-pulmonary  injection 
in  the  human  subject,  we  take  into  account  the  facts  we  have 
discussed,  we  are  justified  in  holding  that  it  is  the  chief  factor  in 
causing  croupous  pneumonia,  and  also  plays  an  important  part 
in  other  forms.  Pneumonia,  in  the  widest  sense  of  the  term,  is, 
however,  not  a  specific  affection,  and  various  inflammatory  con- 
ditions in  the  lungs  can  be  set  up  by  the  different  pyogenic 
organisms,  by  the  bacilli  of  diphtheria,  of  influenza,  etc. 

The  possibility  of  Friedlander's  pneumobacillus  having  an 
etiological  relationship  to  pneumonia  has  been  much  disputed. 
Its  discoverer  found  that  it  was  pathogenic  towards  mice  and 
guinea-pigs,  and  to  a  less  extent  towards  dogs.  Rabbits  appeared 
to  be  immune.  The  type  of  the  disease  was  of  the  nature  of  a 
septicaemia.  No  extended  experiments,  such  as  those  performed 
by  Gamaleia  with  Fraenkel's  coccus,  have  been  done,  and  there- 
fore we  cannot  say  whether  any  similar  pneumonic  effects  are 
produced  by  it  in  partly  susceptible  animals.  The  organism 
appears  to  be  present  alone  in  a  small  number  of  cases  of 
pneumonia,  and  the  fact  that  it  a^so  appears  to  have  been  the 
only  organism  present  in  certain  septic£emic  complications  of 
pneumonia,  such  as  empyema  and  meningitis,  render  it  possible 
that  it  may  be  the  causal  agent  in  a  few  cases  of  the  disease.  It 
is  also  stated  to  have  been  observed  in  certain  cases  of  appen- 
dicitis and  occasionally  in  pysemic  cases. 

In  the  septic  pneumonias  the  different  pyogenic  organisms 
already  described  are  found,  and  sometimes  in  ordinary 
pneumonias,  especially  the  catarrhal  forms,  other  organisms, 
such  as  the  b.  coli  or  its  allies,  may  be  the  causal  agents. 

The  Pathology  of  Pneumococcus  Infection. — The  effects  of 
the  action  of  the  pneumococcus,  at  any  rate  in  a  relatively 
insusceptible  animal  such  as  man,  seem  to  indicate  that  toxins 
may  play  an  important  part.  Pneumonia  is  a  disease  which 
presents  in  many  respects  the  character  of  an  acute  poisoning. 
In  very  few  cases  does  death  take  place  from  the  functions  of 


PNEUMOCOCCUS  INFECTION  239 

the  lungs  being  interfered  with  to  such  an  extent  as  to  cause 
asphyxia.  It  is  from  cardiac  failure,  from  grave  interference 
with  the  heat-regulating  mechanism,  and  from  general  nervous 
depression  that  death  usually  results.  These  considerations, 
taken  in  connection  with  the  fact  that  in  man  the  organisms  are 
found  in  the  greatest  numbers  in  the  lung,  suggest  that  a  toxic 
action  is  at  work.  Various  attempts  have  been  made  to  isolate 
toxins  from  cultures  of  the  pneumococcus,  e.g.,  by  precipitating 
bouillon  cultures  with  alchohol  or  ammonium  sulphate,  and 
poisonous  effects  have  been  produced  by  certain  substances  thus 
derived ;  but  the  effects  produced  are,  as  in  so  many  other 
similar  cases,  of  a  non-specific  character,  and  to  be  classed  as 
interferences  with  general  metabolism.  The  general  conclusion 
has  been  that  the  toxins  at  work  in  pneumonia  are  intracellular ; 
but  no  special  light  has  been  thrown  on  the  common  effects  of 
the  members  of  this  group  of  bacterial  poisons. 

Immunisation  against  the  Pneumococcus. — Animals  can  be 
immunised  against  the  pneumococcus  by  inoculation  with 
cultures  which  have  become  attenuated  by  growth  on  artificial 
media,  or"  with  the  naturally  attenuated  cocci  which  occur  in  the 
sputum  after  the  crisis  of  the  disease.  Netter  effected  immun- 
isation by  injecting  an  emulsion  of  the  dried  spleen  of  .an  animal 
dead  of  pneumococcus  septicaemia.  Virulent  cultures  killed  by 
heating  at  62°  C.,  rusty  sputum  kept  at  60°  C.  for  one  to  two 
hours  and  then  filtered,  and  filtered  or  unfiltered  bouillon 
cultures  similarly  treated  have  also  been  used.  In  all  cases  one 
or  two  injections,  at  intervals  of  several  days,  are  sufficient  for 
immunisation,  but  the  immunity  has  often  been  observed  to 
be  of  a  fleeting  character  and  may  not  last  more  than  a  few 
weeks.  The  serum  of  such  immunised  animals  protects  rabbits 
against  subsequent  inoculation  with  pneumococci,  and  if  injected 
within  twenty-four  hours  after  inoculation,  may  prevent  death. 
A  protective  serum  was  obtained  by  Washbourn,  who  employed 
pneumococcus  cultures  of  constant  virulence.  This  observer 
immunised  a  pony  by  using  successively  (1)  broth  cultures  killed 
by  one  hour's  exposure  to  60°  C. ;  (2)  living  agar  cultures ;  (3) 
living  broth  cultures.  From  this  animal  there  was  obtained  a 
serum  which  protected  susceptible  animals  against  many  times 
an  otherwise  fatal  dose,  and  wThich  also  had  a  limited  curative 
action.  It  is  stated  that  the  serum  of  patients  who  have 
recovered  from  pneumonia  has  in  a  certain  proportion  of  cases 
a  protective  effect  against  the  pneumococcus  in  rabbits,  similar 
to  that  exhibited  by  the  serum  of  immune  animals. 

The  Klemperers  treated  a  certain  number  of  cases  of  human 


240  THE  ACUTE  PNEUMONIAS 

pneumonia  by  serum  derived  from  immune  animals,  and  appar- 
ently with  a  certain  measure  of  success,  and  sera  prepared  by 
Washbourn  and  by  others  have  also  been  used.  The  results 
obtained  by  different  observers  have,  however,  been  rather  con- 
tradictory. The  use  of  these  sera  apparently  causes  the  tempera- 
ture in  some  cases  to.  fall,  and  even  may  hasten  a  crisis,  but 
further  experience  is  necessary  before  their  value  in  therapeutics 
can  be  properly  estimated. 

There  has  been  considerable  difference  of  opinion  as  to  the 
explanations  to  be  given  of  the  facts  observed  regarding  im- 
munisation against  the  pneumococcus  and  especially  regarding 
the  properties  of  immune  sera.  At  first  these  sera  were  supposed 
to  possess  antitoxic  qualities — largely  on  the  ground  that  no 
bactericidal  effect  was  produced  by  them  on  the  bacterium  in 
vitro.  As  no  specific  toxin  has  been  proved  to  be  concerned  in 
the  action  of  the  organism,  the  development  of  an  antitoxin 
during  immunisation  must,  in  the  present  state  of  knowledge, 
be  looked  on  as  not  yet  proved.  To  explain  the  action  of  a 
serum  in  preventing  and  curing  pneumococcal  infections,  it  has 
been  thought  to  have  the  complex  character  seen  in  anti-typhoid 
sera  in  which  two  substances — immune  body  and  complement 
(see  Immunity) — are  concerned,  and  the  variability  in  the 
therapeutic  results  obtained  has  been  accounted  for  on  the  view 
that  there  might  be  a  deficiency  of  complement,  such  as  occurs 
in  other  similar  cases.  The  absence  of  bactericidal  effect,  how- 
ever, raises  several  difficult  points.  It  is  stated  that  no  such 
effect  is  observable  either  in  immune  sera,  or  in  the  serum  of 
patients  who  have  successfully  come  through  an  attack  of  the 
natural  disease.  Some  effect  of  the  kind  would  be  expected  to 
be  present  if  the  anti-pneumonic  serum  were  quite  comparable 
to  the  anti-typhoid  serum.  Within  recent  times  many  have 
turned  to  the  opsonic  property  of  sera  to  account  for  the  facts 
observed.  In  this  connection  Mennes  observed  that  normal 
leucocytes  only  become  phagocytic  towards  pneumococci  when 
they  are  lying  in  the  serum  of  an  animal  immunised  against 
this  bacterium.  Wright  had  in  his  early  papers  looked  to  the 
phagocytosis  of  sensitised  bacteria  to  explain  their  destruction 
in  the  absence  of  bactericidal  qualities  in  the  serum  alone,  and 
Neufeld  and  Rlmpau  have  described  the  occurrence  of  an 
opsonic  effect  in  the  action  of  an  anti-pneumococcic  serum. 
Further  work  may  show  that  along  these  lines  lies  the  explana- 
tion of  the  facts  observed. 

In  studying  further  the  relationship  of  the  opsonic  effect  to 
pneumococcal  infection,  inquiry  has  been  directed  to  the  opsonic 


M  KTHODS  OF  EXAMINATION  241 

qualities  of  the  blood  of  pneumonic  patients,  especially  with  a 
view  to  throwing  light  on  the  nature  of  the  febrile  crisis. 
According  to  some  results,  the  opsonic  index  as  compared  with 
that  of  a  healthy  person  is  not  above  normal,  but  if  the  possible 
phagocytic  capacities  of  the  whole  blood  of  the  sick  person  be 
taken  into  account,  these  will  probably  be  much  above  normal 
in  consequence  of  the  leucocytosis  which  usually  accompanies  a 
successful  resistance  to  this  infection.  It  has  been  observed, 
however,  that  as  the  crisis  approaches  in  a  case  which  is  to 
recover  the  opsonic  index  rises,  and  after  defervescence  gradually 
falls  to  normal.  As  bearing  on  the  factors  involved  in  the 
successful  resistance  of  the  organism  to  the  pneumococcus,  it 
has  been  noted  that  avirulent  pneumococci  are  more  readily 
up^mised  than  more  virulent  strains.  It  is  further  stated  that 
avirulent  cultures  of  the  pneumococcus  can  be  made  to  resist 
phagocytosis  if  they  are  treated  with  the  products  of  the 
autolysis  of  virulent  strains  or  with  washings  from  such  strains, 
and  that  virulent  cocci  if  washed  with  saline  become  capable  of 
1  is -ing  readily  phagocyted.  Further  observations  along  such 
lines  are  to  be  looked  for  with  interest,  and  it  may  be  said  that 
Wright's  vaccination  methods  have  been  applied  to  the  treatment 
of  pneumonic  cases,  and  in  certain  instances  are  said  to  have 
been  followed  by  favourable  result.  It  may  be  noted  here,  in 
conclusion,  that  in  man  it  is  probable  that  immunity  against 
pneumonia  may  be  short-lived,  as  in  a  good  many  cases  of 
pneumonia  a  history  of  a  previous  attack  is  elicited. 

Agglutination  of  the  Pneumococcus. — If  a  small  amount  of  a 
culture  of  Fraenkel's  pneumococcus  be  placed  in  an  anti-pneumo- 
coccic  serum,  an  aggregation  of  the  bacteria  into  clumps  occurs. 
Such  an  agglutination,  as  it  is  called,  is  frequently  observed 
under  similar  circumstances  with  other  bacteria.  The  pheno- 
menon is  not  invariably  associated  with  the  presence  of  protective 
bodies  in  a  serum,  but  it  has  been  used  for  diagnostic  purposes 
in  the  differentiation  of  sore  throats  due  to  pueumococcus  infec- 
tion from  those  due  to  other  bacteria.  Whether  the  method  is 
reliable  has  still  to  be  proved.  It  has  been  shown  that  a  serum 
which  agglutinates  the  pneumococcus  may  also  agglutinate 
streptococci  isolated  from  various  sources.  Such  organisms  are, 
however,  not  so  uniformly  agglutinated  by  a  pneumococcus 
serum  as  are  pneumococci  isolated  from  pneumonic  cases. 

Methods    of    Examination.  —  These    have    been    already 

described,    but   may    be    summarised    thus :    (1)    Microscopic. 

Stain  films  from  the  densest  part  of  the  sputum  or  from  the 

area  of  spreading  inflammation  in  the  lung  by  Grain's  method 

16 


242      EPIDEMIC  CEREBRO-SPINAL  MENINGITIS 

and  by  carbol-fuchsin,  etc.  (pp.  105,  108),  in  the  latter  case 
it  is  usually  best  not  to  decolorise  the  groundwork  of  the 
preparation. 

(2)  By  cultures,  (a)  FraenkeVs  pneumococcus.  With  similar 
material  make  successive  strokes  on  agar,  blood  agar,  or  blood 
serum.  The  most  certain  method,  however,  is  to  inject  some 
of  the  material  containing  the  suspected  cocci  into  a  rabbit.  If 
the  pneumococcus  be  present  the  animal  will  die,  usually  within 
forty -eight  hours,  with  numerous  capsulated  pneumococei  in  its 
heart  blood.  With  the  latter  inoculate  tubes  of  the  above  media 
and  observe  the  growth.  In  some  cases  of  severe  pneumococcic 
infection  the  organism  may  be  cultivated  from  the  blood  obtained 
by  venesection  (p.  72).  (b)  Friedlander 's  pneumohacillus  can 
be  readily  isolated  either  by  ordinary  gelatin  plates  or  by 
successive  strokes  on  agar  media. 

EPIDEMIC  CEKEBRO-SPINAL  MENINGITIS. 

As  the  result  of  observations  on  this  disease  in  different  parts 
of  the  world,  it  has  been  now  established  that  the  causal  agent 

is  the  diplococcus  intra- 
cellularis  meningitidis, 
first  described  by  Weich- 
selbaum,  and  now  often 
known  as  the  meningo- 
coccus.  This  organism  is 
a  small  coccus  measuring 
about  1  /ji  in  diameter 
and  usually  occurs  in 
pairs,  the  adjacent  sides 
being  somewhat  flattened 
against  each  other.  In 
most  cases  the  cocci  are 
chiefly  contained  within 
polymorphonuclear  leuco- 
cytes in  the  exudation 

FIG.  70. — Film  preparation  of  exudation  from     (Fig.  70)  ;   ill  some  cases, 

a  case  of  meningitis  showing -the  diplococci     however)      the      majority 

within  leucocytes.    See  also  Plate  I.,  Fig.  3.  >       .  J        J 

Stained  with  carbol-thionin-blue.      x  1000.        may    be    lying    tree.      It 

stains  readily  with  basic 

aniline  dyes,  but  loses  the  stain  in  Gram's  method.  Both 
in  appearance  and  in  its  staining  reactions  it  is  similar  to 
the  gonococcus  (vide  infra}.  The  organism  can  readily  be 
cultivated  outside  the  body,  but  the  conditions  of  growth 


THE  MENINGOOOOOtfS  243 

are  <  .  >iin-\\  hat  restricted  —  u^ar  with  an  admixture  of  serum 
or  blood  (preferably  human)  is  most  suitable  (p.  43).  Strains 
separated  in  different  epidemics  appear  to  present  slight 
individual  variations,  but  the  following  description  may  be 
taken  as  summing  up  the  common  characters:  —  Growth  takes 
place  best  at  the  temj>erature  of  the  body,  and  practically 
(•t-ases  at  25°  C.  On  serum  agar  the  colonies  are  circular  discs 
of  somewhat  transparent  appearance,  and  possessing  a  smooth, 
shining  surface  ;  they  have  little  tendency  to  become  confluent. 
When  examined  under  a  low  magnification  the  colour  is  seen  to 
be  somruliat  yellowish,  and  the  margins  usually  are  smooth 
and  regular,  though  on 

some  media  slight  crena-  •  "  ^ 

tion   may   appear.     The  '-.*** 

colonies  may  be  of  con-  9          *•  % 

siderable    size,    reaching  •*'     .          "'*""*  **t,  » 

sometimes  a  diameter  of  *    "     *  ./,  *  ».        »*k 

2  to  3  mm.  on  the  third       *  ."  '      ' 


day.     On  plain  agar  the    *t 

colonies   are   very   niudi  •      *    j£%  **  ' 

smaller,   and    sometimes  * 

no  growth  occurs;    sub-      - 

cultures  esi>ecially  often 

fail  to  give  any  growth  •       •    •  „*  ^  *     •     •  t 

on    this     medium.       In  N  *  ^    *.    j      .. 

serum  bouillon  the  organ-  •/"•*«•  >  *"• 

ism    ]>roduces  a  general 

turbidity  with  formation     Vu;.  71.—  Pure.  culture  of  <liplococcus  intra- 

of   some  deposit    after    a  '-fllulsiris,  sliowiiiK  involution  forms. 

day  or  two.      It  ferments 

maltose  and  dextrose  with   acid  production,  a  pro}>erty  which 

distinguishes  it  from  the  micrococcus  catarrhalis  (vide  infra). 

Fermentation  tests  are  most  satisfactorily  carried  out  by  means 

of  solid  serum  media  containing  1'  per  cent,  of  the  sugar  to  be 

tested    (p.  80).      In   all   cases   growth   occurs   best  when   the 

medium   ha-  a  neutral  or  very  slightly  alkaline   reaction.     In 

cultures  the  organism  presents  the  same  appearance  as  in  the 

body,  and  often  shows  tetrad  formation.     There  is  also  a  great 

tendency  to  the  production  of  involution  forms  (Fig.  71),  many 

of  the  cocci  becoming  much  swollen,  staining  badly,  and  after- 

wards  undergoing   disintegration.     This   change,   according  to 

Flexner's  observations,  would  api>ear  to  be  due  to  the  production 

of  an  autolytic  enzyme,  and  he  has  also  found  that  this  substance 

has  the  property  of  producing  dissolution  of  the  bodies  of  other 


244      EPIDEMIC  CEREBRO-SPINAL  MENINGITIS 

bacteria.  The  life  of  the  organism  in  cultures  is  a  comparatively 
short  one ;  after  a  few  days  cultures  will  often  be  found  to  be 
dead,  but,  by  making  sub-cultures  every  three  or  four  days, 
strains  can  be  maintained  alive  for  considerable  periods.  The 
organism  is  readily  killed  by  heat  at  60°  C.,  and  it  is  also  very 
sensitive  to  weak  antiseptics ;  drying  for  a  period  of  a  day  has 
been  found  to  be  fatal  to  it.  The  facts  established  accordingly 
show  it  to  be  a  somewhat  delicate  parasite. 

As  stated  above,  the  organism  occurs  in  the  exudate  in  the 
meninges  and  in  the  cerebro-spinal  fluid,  and  it  can  usually  be 
obtained  by  lumbar  puncture.  In  acute  cases,  especially  in  the 
earlier  stages,  it  is  usually  abundant ;  but  in  the  later  stages  of 
cases  of  more  sub-acute  character,  its  detection  may  be  a  matter 
of  difficulty,  and  only  a  few  examples  may  be  found  after  a 
prolonged  search ;  in  extremely  acute  cases  also  the  organism 
may  be  difficult  to  demonstrate.  In  most  cases  the  disease  is 
practically  restricted  to  the  nervous  system,  but  occasionally 
complications  occur,  and  in  these  the  organism  may  sometimes 
be  found.  It  has  been  observed,  for  example,  in  arthritis,  peri- 
carditis, pneumonic  patches  in  the  lung,  and  in  other  inflam- 
matory conditions  associated  with  the  disease.  In  a  certain 
number  of  cases  it  has  been  obtained  from  the  blood  during  life, 
but  cultures  in  most  instances  give  negative  results. 

Experimental  inoculation  shows  that  the  ordinary  laboratory 
animals  are  relatively  insusceptible  to  this  organism.  An  in- 
flammatory condition  may  be  produced  in  mice  and  guinea-pigs 
by  intra-peritoneal  injection,  and  a  fatal  result  may  follow ;  in 
such  cases  the  organism  does  not  seem  to  undergo  very  active 
multiplication,  though  it  may  sometimes  be  cultivated  from  the 
blood,  and  none  of  the  lesions  in  the  nervous  system  are  repro- 
duced. Flexner  and  Stuart  M  'Donald  have  shown  that  cerebro- 
spinal  meningitis  may  be  produced  in  monkeys  by  injections  of 
the  organism  into  the  spinal  canal,  the  latter  observer  finding 
that  exudate  containing  mehingococci  was  more  effective  than 
cultures.  In  such  experiments  the  organism  extends  upwards 
to  the  brain,  and  produces  meningitis  within  a  very  short  time. 
The  resulting  lesions,  both  as  regards  their  distribution  and 
general  characters,  and  also  as  regards  the  histological  changes, 
resemble  the  disease  in  the  human  subject.  Even  these  animals, 
however,  are  manifestly  less  susceptible  than  the  human 
subject. 

Many  questions  of  great  importance  with  regard  to  the  spread 
of  the  disease  still  require  further  investigation.  The  organism 
has  been  obtained  by  culture  from  the  throat  and  nasal  cavities 


SERUM  REACTIONS  245 

of  those  suffering  from  the  disease  in  a  considerable  number  of 
instances.  It  has  also  been  obtained  from  the  same  positions  in 
healthy  individuals,  during  an  epidemic  of  the  disease.  In 
some  epidemics  also  a  pharyngitis  has  been  found  to  occur,  and 
the  organism  has  been  obtained  from  the  affected  fauces.  The 
majority  of  workers  at  this  subject  are  inclined  to  believe  that 
the  organism  spreads  by  means  of  the  lymphatics  from  the 
pharynx  or  nose  to  the  base  of  the  brain,  but  direct  evidence 
that  this  occurs  has  not  been  supplied.  On  the  other  hand,  the 
facts  established  with  regard  to  other  infections  make  it  quite 
probable  that  the  organism  gains  entrance  to  the  blood  stream 
from  the  upper  respiratory  passages,  and  then  settles  in  the 
meninges.  Infection  by  the  alimentary  canal,  the  organisms 
thereafter  reaching  the  spinal  meninges  by  the  lymphatics,  has 
been  suggested  as  a  possibility,  but  such  a  view  does  not  appear 
to  have  much  support.  Whatever  may  be  found  to  be  the  path 
by  which  the  organism  reaches  the  brain,  the  evidence  at 
present  tends  to  show  that  the  entrance  of  the  organism  into 
the  body  is  by  the  naso-pharynx,  and  that  this  usually  results 
by  inhalation  of  the  organism  distributed  in  fine  particles  of 
expectoration,  etc.  In  fact,  as  regards  the  mode  and  conditions 
of  infection,  an  analogy  would  appear  to  hold  between  this 
disease  and  influenza. 

Apart  from  the  epidemic  form  of  the  disease,  cases  of  sporadic 
nature  also  occur,  in  which  the  lesions  are  of  the  same  nature, 
and  in  which  the  meningococcus  is  present.  The  facts  stated 
would  indicate  that  the  origin  and  spread  of  the  disease  in  the 
epidemic  form  depend  on  certain  conditions  which  produce  an 
increased  virulence  of  the  organism.  We  are,  however,  as  yet 
entirely  ignorant  as  to  what  these  conditions  may  be.  In 
simple  posterior  basal  meningitis  in  children  a  diplococcus  is 
present,  as  described  by  Still,  which  has  the  same  microscopic 
and  cultural  characters  as  the  diplococcus  intracellularis ;  it  has 
been  regarded  as  probably  an  attenuated  variety  of  the  latter. 
Recently,  however,  Houston  and  Ran  kin  have  found  that  the 
serum  of  a  patient  suffering  from  epidemic  meningitis  does  not 
exert  the  same  opsonic  and  agglutinative  effects  on  the  diplo- 
coccus of  basal  meningitis  as  on  the  diplococcus  intracellularis ; 
and  this  result  points  to  the  two  organisms  being  distinct, 
though  closely  allied,  species. 

Serum  Reactions. — An  agglutination  reaction  towards  the 
•  lijiplococcus  intracellularis  is  given  by  the  serum  of  patients 
suffering  from  the  disease,  where  life  is  prolonged  for  a  sufficient 
length  of  time.  It  usually  appears  about  the  fourth  day,  when 


246     EPIDEMIC  CEREBRO-SPINAL  MENINGITIS 

the  serum  may  give  a  positive  reaction  in  a  dilution  of  1  :  50 ; 
at  a  later  stage  it  lias  been  observed  in  so  great  a  dilution  as 
1  :  1000.  Specific  opsonins  may  appear  in  the  blood  about 
the  same  time,  and  though  they  are  not  always  proportional  in 
amount  to  the  agglutinins,  the  two  classes  of  substances  have 
pretty  much  the  same  significance,  and  may  occasionally  be  of 
use  in  diagnosis  when  lumbar  puncture  fails  to  give  positive 
results.  Although  their  presence  in  large  amounts  may  be  said 
to  indicate  a  marked  reaction,  they  do  not  supply  information  of 
much  value  in  relation  to  prognosis.  Immune-bodies,  as  shown 
by  bactericidal  and  deviation  of  complement  tests  (p.  126,  130), 
may  also  be  developed  in  considerable  amount  in  the  course  of 
the  disease. 

Anti-sera  for  therapeutical  purposes  have  been  introduced  by 
various  workers,  and  of  these  the  one  which  has  been  most 
extensively  used  is  that  of  Flexner  and  Jobling.  This  serum 
is  prepared  from  the  horse  by  repeated  injections  in  increasing 
doses  of  dead  cultures,  followed  by  injections  of  culture  autolysate 
and  of  living  cultures,  these  two  latter  being  best  administered 
by  the  subcutaneous  method.  Several  strains  of  meningococci 
are  mixed  together  for  purposes  of  injection,  and  the  immunisa- 
tion is  continued  over  a  period  of  several  months.  For  treat- 
ment of  the  disease  the  serum  is  injected  under  the  spinal 
dura,  30  c.c.  being  generally  used  for  an  injection,  this  being 
continued  for  several  days.  This  serum  has  been  used  on  a 
large  scale  in  various  parts  of  the  world,  and  there  is  general 
agreement  as  to  its  favourable  effects — the  mortality  of  the 
disease,  which  is  generally  70  to  80  per  cent.,  having  been 
reduced  to  about  30  per  cent,  or  even  less.  By  means 
of  its  use  the  tendency  to  the  occurrence  of  chronic  lesions 
has  also  been  markedly  diminished.  The  action  of  such 
anti-sera  cannot  as  yet  be  fully  explained.  They  certainly 
contain  opsouins,  agglutinins,  immune-bodies  which  bind  com- 
plement, and  possibly  also  anti-endotoxins.  After  the  injection 
the  number  of  meningococci  becomes  markedly  reduced, 
probably  as  a  result  of  increased  phagocytosis;  there  can 
scarcely  be  any  direct  bactericidal  action  owing  to  the  absence 
of  complement.  The  standardisation  of  such  anti-sera  is  a 
matter  of  some  difficulty ;  at  first  the  deviation  of  complement 
method  was  used  (p.  130),  but  now  the  opsonic  index  is  regarded 
with  more  favour  as  an  index  of  the  potency  of  the  serum. 
Mackenzie  and  Martin  have  treated  cases  by  the  intra-spinal 
injection  of  the  fresh  serum  of  patients  suffering  from  the 
disease  or  who  have  recovered  from  it,  such  serum  being  in 


ALLIED  DIPLOCOCCI  247 

many  cases  rich  in  immune-bodies  for  the  meningococcus,  and 
possessing  a  greatly  increased  bactericidal  action  as  compared 
with  normal  serum.  Though  the  number  of  cases  treated  by 
this  method  is  not  yet  large,  a  distinctly  favourable  result  has 
been  obtained. 

Allied  Diplococci. — In  the  naso-pharynx  there  occur  other 
Gram-negative  diplococci  which  have  j  a  close  resemblance  to 
the  diplococcus  intracellularis.  These  occur  in  the  healthy 
state,  but  are  especially  abundant  in  catarrhal  conditions.  Of 
these  the  diplococcus  catarrhalis  has  the  closest  resemblance  to 
the  diplococcus  intracellularis.  In  addition  to  occurring  in 
health  this  organism  has  also  been  found  in  large  numbers  in 
•epidemic  catarrh.  Its  microscopic  appearances  are  practically 
similar  to  those  described  above,  and  it  also  occurs  within  leuco- 
cytes. Its  colonies  on  serum  agar  are  more  opaque  than  those 
of  the  diplococcus  intracellularis,  and  often  have  a  somewhat 
firm  though  friable  consistence,  so  that  they  are  sometimes 
removed  en  masse  by  the  platinum  needle.  The  organism  grows 
on  gelatin  at  20°  C.  without  liquefying  the  medium,  and  it  has 
none  of  the  fermentative  properties  described  above  as  belonging 
to  the  diplococcus  intracellularis.  The  diplococcus  pharyngis 
mccus  (v.  Lingelsheim)  also  grows  at  room  temperature,  and  its 
colonies  are  very  tough  and  adhere  to  the  surface  of  the  medium ; 
it  can  thus  readily  be  distinguished  from  the  meningococcus.  It 
has  marked  fermentative  properties,  acting  on  glucose,  maltose, 
saccharose,  and  laevulose.  The  diplococcus  mucosus  has  colonies 
of  slimy  consistence ;  it  grows  at  room  temperature,  and  it  forms 
capsules,  which  can  be  demonstrated  by  the  method  of  Hiss. 
There  are  other  Gram-negative  diplococci  which  are  chromogenic, 
and  thus  can  readily  be  distinguished.  The  points  of  difference 
between  the  meningococcus  and  the  gonococcus  are  given  on 
p.  252.  A  Gram-positive  diplococcus  called  the  diplococcus 
crassus  is  also  of  common  occurrence;  it  is  rather  larger  than 
the  diplococcus  intracellularis,  and  especially  in  sub-cultures  may 
tend  to  assume  staphylococcal  forms. 

Apart  from  the  epidemic  form  of  the  disease,  meningitis  may 
be  produced  by  almost  any  of  the  organisms  described  in  the 
previous  chapter,  as  associated  with  inflammatory  conditions. 
A  considerable  number  of  cases,  especially  in  children,  are  due 
to  the  pneumococcus.  In  many  instances  where  no  other  lesions 
are  present  the  extension  is  by  the  Eustachian  tube  to  the  middle 
ear.  In  other  cases  the  path  of  infection  is  from  some  other 
lesion  by  means  of  the  blood  stream.  This  organism  also  infects 
the  meninges  not  infrequently  in  lobar  pneumonia,  and  in  some 


248     EPIDEMIC  CEREBRO-SPINAL  MENINGITIS 

cases  with  head  symptoms  we  have  found  it  present  where  there 
was  merely  a  condition  of  congestion.  The  pneumobacillus  also- 
has  been  found  in  a  few  cases.  Meningitis  is  not  infrequently 
produced  by  streptococci,  especially  when  middle-ear  disease  is 
present,  less  frequently  by  one  of  the  staphylococci ;  occasionally 
more  than  ;  one  organism  may  be  concerned.  In  meningitis 
following  influenza  the  influenza  bacillus  has  been  found  in  a 
few  instances,  but  sometimes  the  pneumococcus  is  the  causal 
agent.  Cases  of  meningitis  occur  associated  with  organisms 
which  resemble  the  influenza  bacillus  morphologically  and  also 
in  presenting  haemophilic  culture  reactions,  but  which  possess 
pathogenic  properties  for  rabbits  and  guinea-pigs.  These  bacilli 
frequently  both  in  the  cerebro-spinal  fluid  and  in  cultures  show 
a  tendency  to  produce  long  filamentous  forms  and  also  under 
both  circumstances  may  show  a  beading  of  the  protoplasm 
which  gives  them  a  diphtheroid  appearance.  Gram-negative 
anaerobic  bacilli  have  also  been  found  in  cases  of  meningitis. 
An  invasion  of  the  meninges  by  the  anthrax  bacillus  occurs, 
but  is  a  rare  condition ;  it  is  attended  by  diffuse 
haemorrhage  in  the  subarachnoid  space.  In  tubercular 
meningitis  the  tubercle  bacillus,  of  course,  is  present,  especially 
in  the  nodules  along  the  sheaths  of  the  vessels. 

In  conclusion,  here  it  may  be  stated  that  mixed  infections 
may  occur  in  meningitis.  Thus  the  pneumococcus  has  been 
found  associated  with  the  tubercle  bacillus  and  also  with  the 
diplococcus  intracellularis. 

Methods  of  Examination. — During  life  these  involve  the  microscopic 
investigation  of  the  centrifuged  lumbar  puncture  fluid  and  making 
cultures  therefrom.  For  the  former,  Gram-stained  smears  make  the 
recognition  of  the  meningococcus  relatively  easy,  and  the  presence  of 
Gram-negative  cocci,  especially  within  cells,  is  practically  diagnostic  of 
a  case  of  cerebro-spinal  fever.  Tubes  of  serum-agar,  nasgar  (pp.  42,  43), 
or  agar  containing  25  per  cent,  of  ascitic  or  ovarian  fluid,  may  then  be 
inoculated.  The  difficult  cases  are  those  where  no  bacteria  can  be  found 
microscopically  in  the  lumbar  fluid.  Here  the  character  of  the  exudate 
may  give  help.  A  predominance  of  polymorphonuclear  cells  is  usually 
manifest  in  meningococcic,  pneumococcic,  and  influenzal  cases,  whereas  in 
tubercular  meningitis  the  exudate  is  chiefly  lymphocytic.  In  such 
circumstances,  besides  other  media,  a  tube  of  blood-smeared  agar  should 
be  inoculated  in  case  the  pneumococcus  or  the  influenza  bacillus  is  the 
causal  organism.  To  speak  generally,  if  with  a  polymorphonuclear  exu- 
date no  growth  occurs  in  the  media  mentioned  the  case  is  most  likely  to  be 
due  to  the  meningococcus.  In  tubercular  cases  it  is  sometimes  impossible 
to  demonstrate  the  bacilli  microscopically  in  the  exudate,  though  on 
careful  search  they  may  often  be  found. 


CHAPTER  IX. 

GONORRHOEA  AND  SOFT  SORE. 

GONORRH(EA. 

Introductory. — The  micrococcus  now  known  to  be  the  cause 
of  gonorrhoea,  and  called  the  gonococcus,  was  first  described 
by  Neisser,  who  in  1879  gave  an  account  of  its  microscopical 
characters  as  seen  in  the  pus  of  gonorrhceal  affections,  both  of 
the  urethra  and  of  the  conjunctiva.  He  considered  that  this 
organism  was  peculiar  to  the  disease,  and  that  its  characters 
were  distinctive.  Later  it  was  successfully  isolated  and  cultivated 
on  solidified  human  serum  by  Bumm  and  others.  Its  characters 
have  since  been  minutely  studied,  and  by  inoculations  of  cultures 
on  the  human  subject  its  causal  relationship  to  the  disease  has 
been  conclusively  established. 

The  Gonococcus. — Microscopical  Characters. — The  organism 
of  gonorrhoea  is  a  small  micrococcus  which  usually  is  seen  in  the 
diplococcus  form,  the  adjacent  margins  of  the  two  cocci  being 
flattened,  or  even  slightly  concave,  so  that  between  them  there  is 
a  small  oval  interval  which  does  not  stain.  An  appearance  is 
thus  presented  which  has  been  compared  to  that  of  two  beans 
placed  side  by  side  (vide  Fig.  72).  When  division  takes  place  in 
the  two  members  of  a  diplococcus,  a  tetrad  is  formed,  which, 
however,  soon  separates  into  two  sets  of  diplococci — that  is  to 
say,  arrangement  as  diplococci  is  much  commoner  than  as  tetrads. 
Cocci  in  process  of  degeneration  are  seen  as  spherical  elements 
of  varying  size,  some  being  considerably  swollen. 

These  organisms  are  found  in  large  numbers  in  the  pus  of 
acute  gonorrhoea,  both  in  the  male  and  female,  and  for  the  most 
part  are  contained  within  the  leucocytes.  In  the  earliest  stage, 
when  the  secretion  is  glairy,  a  considerable  number  are  lying 
free,  or  are  adhering  to  the  surface  of  desquamated  epithelial 
cells,  but  when  it  becomes  purulent  the  large  proportion  within 
eucocytes  is  a  very  striking  feature.  In  the  leucocytes  they  He 

249 


250 


GONORRHCEA  AND  SOFT  SORE 


within  the  protoplasm,  especially  superficially,  and  are  often  so 
numerous  that  the  leucocytes  appear  to  be  filled  with  them,  and 
their  nuclei  are  obscured.  As  the  disease  becomes  more  chronic, 

the  gonococci  gradually 
become  diminished  in 
number,  though  even  in 
long-standing  cases  they 
may  still  be  found  in  con- 
siderable numbers.  They 
are  also  present  in  the 
purulent  secretion  of 
gonorrhoeal  conjunctivitis, 
also  in  various  parts  of 
the  female  genital  organs 
when  these  parts  are  the 
seat  of  true  gonorrhreal 
infection,  and  they  have 
been  found  in  some  cases 
in  the  secondary  infections 
of  the  joints  in  the  dis- 
ease, as  will  be  described 
below. 

Stained  with  fuchsin.     x  1000.  Staining. —  The     gono- 

coccus  stains   readily  and 

deeply  with  a  watery  solution  of  any  of  the  basic  aniline  dyes — 
methylene-blue,  fuchsin,  etc.  It  is,  however,  easily  decolorised, 
and  it  completely  loses  the  stain  by  Gram's  method — an 
important  point  in  the  microscopical  examination. 

Cultivation  of  the  Gonococcus.  —  This  is  attended  with 
some  difficulty,  as  the  conditions  of  growth  are  somewhat 
restricted.  The  most  suitable  media  are  "blood-agar"  and -the 
serum  media  already  described  for  the  purpose  (pp.  42,  43).  It 
is  advisable  to  inoculate  the  media  within  half  an  hour  after 
obtaining  the  material  from  the  body,  and  place  the  tubes 
at  once  in  the  incubator.  Growth  takes  place  best  at  the 
temperature  of  the  body,  and  ceases  altogether  at  25°  C.  Cultures 
are  obtained  by  taking  some  pus  on  the  loop  of  the  platinum 
needle  and  inoculating  one  of  the  media  mentioned  by  leaving 
minute  quantities  here  and  there  on  the  surface.  The  medium 
may  be  used  either  as  ordinary  "  sloped  tubes  "  or  as  a  thin  layer 
in  a  Petri's  capsule.  The  young  colonies  are  usually  visible 
within  forty-eight  hours,  and  often  within  twenty-four  hours ; 
it  is  important,  however,  to  note  that  sometimes  growth  may 
not  appear  till  the  fourth  day.  They  appear  around  the 


FIG.  72. — Portion  of  film  of  gonorrhoeal  pus, 
showing  the  characteristic  arrangement  of 
the  gonococci  within  leucocytes.  See  also 
Plate  I.,  Fig.  5. 
d  wit 


CULTIVATION  OF  GONOCOCCUS 


251 


points  of  inoculation  as  small  semi-transparent  discs  of  rounded 
shape.      The    colonies  vary   somewhat   in    size,    and    tend    to 


#•  ft- 


.*-v  \ 


FIG.  73. — Colonies  of  gonococcus  on  serum  agar  ;  (a)  three  days'  growth  ; 

(6)  and  (c)  five  days'  growth,      x  9. 
From  photographs  l.y  Dr.  W.  B.  M.  Martin. 

remain  more  or  less  separate.  Later,  the  margin  tends  to  be 
undulated  and  the  centre  more  opaque  ;  a  radial  marking  may 
be  present  (Fig.  73).  The  first  cultures  die  out  somewhat 

•  I  u  irk  ly,      but      in     sub- 
cultures, kept  at   37°  C., 
the      organism      remains 
alive    for    a    considerable 
time,      sometimes      three       /g 
weeks.       After     a     week     [M 
more  active  foci  of  growth     /  |- 
may   appear    in    some    of 

tin-  colonies  in  the  form    j 

of      heaped -up      opaque    \£g 

points,     thus    giving     an 

appearance    suggestive  of 

contamination.       In     the 

early  stage  of  the  disease 

the    organism    is    present 

in    the   male    urethra    in    Fj(;  74._Gonococci;  from  a  pure  culture 

practically  pure  condition,       On   blood    agar   of  twenty-four    hours' 

and  if  the  meatus  of  the        growth.     Some  already  are  beginning  to 

urethra    be    sterilised    by        ^owth^swojlen  appearance  cominon  in 

washing    with    weak    solu-     Stained  with  carbol-thionin  blue,     x  1000. 
t  ion  of  corrosive  sublimate 

and  then  with  absolute  alcohol,  and  the  material  for  inoculation 
be  expressed  from  the  deeper  part  of  the  urethra,  cultures  may 

•  •ft en  be  obtained  which  are  pure  from  the  first.     In  culture  the 


252  GONORRHOEA  AND  SOFT  SORE 

organisms  have  similar  microscopic  characters  to  those  described 
(Fig.  74),  but  show  a  remarkable  tendency  to  undergo  degenera- 
tion, becoming  swollen  and  of  various  sizes,  and  staining  very 
irregularly.  Degenerated  forms  are  seen  even  on  the  second 
day,  whilst  in  a  culture  four  or  five  days  old  comparatively  few 
normal  cocci  may  be  found.  The  less  suitable  the  medium  the 
more  rapidly  does  degeneration  take  place.  When  mixed  with 
other  organisms  the  gonococcus  may  be  separated  by  serum  agar 
plates  (p.  43). 

On  ordinary  agar  and  on  glycerin-agar  some  growth  may 
take  place  when  the  reaction  is  just  alkaline  to  litmus,  but  these 
media  are  quite  unsuitable  for  ordinary  purposes.  The  organism 
does  not  grow  on  gelatin,  potato,  etc. 

Comparison  with  Meningococcus. — The  morphological  and 
cultural  characters  of  the  gonococcus  and  meningococcus  are 
in  many  respect  closely  similar;  the  following  points  are  of 
importance  in  distinguishing  them.  The  conditions  of  growth 
of  the  gonococcus  are  more  restricted  than  those  of  the  meningo- 
coccus. The  gonococcus  usually  does  not  grow  on  the  ordinary 
agar  media,  whereas  the  meningococcus  grows  well,  at  least 
after  the  first  sub-culture.  The  colonies  of  the  latter  are  more 
opaque  and  have  more  regular  margins  than  those  of  the 
gonococcus.  The  meningococcus  grows  well  in  bouillon,  pro- 
ducing a  general  turbidity,  whereas  the  gonococcus  does  not 
grow ;  even  in  serum  bouillon  the  latter  organism  flourishes 
feebly,  and  the  scanty  growth  falls  to  the  bottom  leaving  the 
medium  clear,  whilst  the  meningococcus  produces  abundant 
growth  with  general  turbidity.  The  fermentative  effects  have 
also  been  studied,  and  the  chief  results  obtained  are  that  glucose 
is  the  only  sugar  usually  employed  which  is  fermented  by  the 
gonococcus,  whereas  the  meningococcus  always  ferments  maltose 
also.  (For  fermentative  tests  in  the  case  of  the  gonococcus, 
solid  media,  as  introduced  by  v.  Lingelsheim,  should  be  used, 
the  serum  medium  of  Martin,  with  litmus  and  the  particular 
sugar  added,  being  specially  suitable.) 

Specific  serum  reactions  —  agglutination,  opsonic  action, 
bactericidal  action,  and  fixation  of  complement — have  been 
studied  by  Torrey,  Elser  and  Huntoon,  and  Martin,  in  the  case 
of  the  two  organisms.  The  general  results  obtained  are  that 
each  organism  represents  a  somewhat  heterogeneous  group 
showing  considerable  variations  as  regards  the  tests  mentioned. 
An  anti-gonococcus  serum  produced  by  injecting  one  strain  of 
gonococcus  has  the  maximum  effect  on  that  strain,  whilst  its 
Action  on  other  strains  may  be  much  feebler  •  so  also  with  an 


RELATIONS  TO  THE  DISEASE  253 

aiiti-meuingococciis  serum  in  relation  to  different  strains  of 
mt'iiingococci.  An  anti-gonococcus  serum  may  have  some  effect, 
though  usually  very  slight,  on  a  meningococcus  and  vice  versa ; 
this  indicates  that  there  are  some  receptors  common  to  the  two 
organisms.  These  results  are  in  many  ways  comparable  with 
the  facts  established  with  regard  to  members  of  the  typhoid-coli 
group,  and  are  of  course  quite  compatible  with  the  gonococcus 
and  the  meniugococcus  being  distinct  species. 

Relations  to  the  Disease. — The  gouococcus  is  invariably 
present  in  the  urethral  discharge  in  gonorrhoea,  and  also  in 
other  parts  of  the  genital  tract  when  these  are  the  seat  of  true 
gonorrhoeal  infection.  Its  presence  in  these  different  positions 
has  been  demonstrated  not  only  by  microscopic  examination 
but  also  by  culture.  From  the  description  of  the  conditions  of 
growth  in  culture  it  will  be  seen  that  a  life  outside  the  body 
in  natural  conditions  is  practically  impossible — a  statement 
which  corresponds  with  the  clinical  fact  that  the  disease  is 
always  transmitted  directly  by  contagion.  Inoculations  of  pure 
cultures  on  the  urethra  of  lower  animals,  and  even  of  apes,  is 
followed  by  no  effect,  but  a  similar  statement  can  be  made  with 
regard  to  inoculations  of  gonorrhceal  pus  itself.  In  fact, 
hitherto  it  has  been  found  impossible  to  reproduce  the  disease  by 
any  means  in  the  lower  animals.  On  a  considerable  number  of 
occasions  inoculations  of  pure  cultures  have  been  made  on  the 
human  urethra,  both  in  the  male  and  female,  and  the  disease, 
with  all  its  characteristic  symptoms,  has  resulted.  (Such 
experiments  have  been  performed  independently  by  Bumm, 
Steinschneider,  Wertheim,  and  others.)  The  causal  relationship 
of  the  organism  to  the  disease  has  therefore  been  completely 
established,  and  it  is  interesting  to  note  how  the  conditions  of 
growth  and  the  pathogenic  effects  of  the  organism  agree  with 
the  characters  of  the  natural  disease. 

I n tra peritoneal  injections  of  pure  cultures  of  the  gonococcus  in  white 
mice  produce  a  localised  peritonitis  with  a  small  amount  of  suppuration, 
the  organisms  being  found  in  large  numbers  in  the  leucocytes  (Wertheim). 
They  also  penetrate  the  peritoneal  lining  and  are  found  in  the  sub- 
eudothelial  connective  tissue,  but  they  appear  to  have  little  power  of 
proliferation,  they  soon  disappear,  and  the  inflammatory  condition  does 
not  spread.  Injection  of  pure  cultures  into  the  joints  of  rabbits,  dogs, 
and  guinea-pigs  causes  an  acute  inflammation,  which,  however,  soon 
subsides,  whilst  the  gonococci  rapidly  die  out  ;  a  practically  similar 
result  is  obtained  when  dead  cultures  are  used.  These  experiments  show 
that  while  the  organism,  when  present  in  large  numbers,  can  produce  a 
certain  amount  of  inflammatory  change  in  these  animals,  it  has  little  or 
no  power  of  multiplying  and  spreading  in  their  tissues. 

Toxin  of  the  Gonococcus.— De  Christmas  has  cultivated  the  gonococcus 


254  GONORRHOEA  AND  SOFT  SORE 

in  a  mixture  of  one  part  of  ascitic  fluid  and  three  parts  of  bouillon,  and 
has  found  that  the  fluid  after  twelve  days'  growth  has  toxic  properties. 
At  this  period  all  the  organisms  are  dead  ;  such  a  fluid  constitutes  the 
"toxin."  The  toxic  substances  are  precipitated  along  witli  the  proteids 
by  alcohol,  and  the  precipitate  after  being  desiccated  possesses  the  toxic 
action.  In  young  rabbits  injection  of  the  toxin  produces  suppuration  ; 
this  is  well  seen  in  the  anterior  chamber  of  the  eye,  where  hypopyon 
results.  The  most  interesting  point,  however,  is  with  regard  to  its 
action  on  mucous  surfaces  ;  for,  while  in  the  case  of  animals  it  produces 
no  effect,  its  introduction  into  the  human  urethra  causes  acute  catarrh, 
attended  with  purulent  discharge.  He  found  that  no  tolerance  to  the 
toxin  resulted  after  five  successive  injections  at  intervals.  In  a  more 
recent  publication  he  points  out  that  the  toxin  on  intracerebral  injection 
has  marked  effects  ;  he  also  claims  to  have  produced  an  antitoxin. 
He  states  that  the  toxin  diffuses  out  in  the  culture  medium,  and  does 
not  merely  result  from  disintegration  of  the  organisms.  This  has, 
however,  been  called  in  question  by  other  investigators. 

Distribution  in  the  Tissues. — The  gonococcus  having  been 
thus  shown  to  be  the  direct  cause  of  the  disease,  some  additional 
facts  may  be  given  regarding  its  presence  both  in  the  primary 
and  secondary  lesions.  In  the  human  urethra  the  gonococci 
penetrate  the  mucous  membrane,  passing  chiefly  between  the 
epithelial  cells,  causing  a  loosening  and  desquamation  of  many 
of  the  latter  and  inflammatory  reaction  in  the  tissues  below, 
attended  with  great  increase  of  secretion.  There  occurs  also 
a  gradually  increasing  emigration  of  leucocytes,  which  take  up 
a  large  number  of  the  organisms.  The  organisms  also  penetrate 
the  subjacent  connective  tissue  and  are  especially  found,  along 
with  extensive  leucocytic  emigration,  around  the  lacunae.  Here 
also  many  are  contained  within  leucocytes.  Even,  however, 
when  the  gonococci  have  disappeared  from  the  urethral  dis- 
charge, they  may  still  be  present  in  the  deeper  part  of  the 
mucous  membrane  of  the  urethra,  possibly  also  in  the  prostate, 
and  may  thus  be  capable  of  producing  infection.  The  prostatic 
secretion  may  sometimes  be  examined  by  making  pressure  on 
the  prostate  from  the  rectum  when  the  patient  has  almost 
emptied  his  bladder,  the  secretion  being  afterwards  discharged 
along  with  the  remaining  urine.  (Foulerton.)  In  acute 
gonorrhoea  there  is  often  a  considerable  degree  of  inflammatory 
affection  of  the  prostate  and  vesiculae  seminales,  but  whether 
these  conditions  are  always  due  to  the  presence  of  gonococci 
in  the  affected  parts  we  have  not  at  present  the  data  for  deter- 
mining. A  similar  statement  also  applies  to  the  occurrence  of 
orchitis  and  also  of  cystitis  in  the  early  stage  of  gonorrhoea. 
Gonococci  have,  however,  been  obtained  in  pure  culture  from 
peri-urethral  abscess  and  from  epididymitis  :  it  is  likely  that 


DISTRIBUTION  OF  GONOCOCCUS  255 

the  latter  condition,  when  occurring  in  gonorrhoea,  is  usually 
due  to  the  actual  presence  of  gonococci.  During  the  more 
chronic  stages  other  organisms  may  appear  in  the  urethra,  aid 
in  maintaining  the  irritation,  and  may  produce  some  of  the 
secondary  results.  The  bacillus  coli,  the  pyogenic  cocci,  etc., 
are  often  present,  and  may  extend  along  the  urethra  to  the 
bladder  and  set  up  cystitis,  though  in  .this  they  may  be  aided 
by  the  passage  of  a  catheter.  It  may  be  mentioned  here  that 
Wertheim  cultivated  the  gonococcus  from  a  case  of  chronic 
gonorrhea  of  two  years'  standing,  and  by  inoculation  on  the 
human  subject  proved  it  to  be  still  virulent. 

In  the  disease  in  the  female,  gonococci  are  almost  invariably 
present  in  the  urethra,  the  situation  affected  next  in  frequency 
being  the  cervix  uteri.  They  do  not  appear  to  infect  the  lining 
epithelium  of  the  vagina  of  the  adult  unless  some  other  abnormal 
condition  be  present,  but  they  do  so  in  the  gonorrhceal  vulvo- 
vaginitis  of  young  subjects.  They  have  also  been  found  in 
suppurations  in  connection  with  Bartholini's  glands,  and  some- 
times produce  an  inflammatory  condition  of  the  mucous 
membrane  of  the  body  of  the  uterus.  They  may  also  pas.- 
along  the  Fallopian  tubes  and  produce  inflammation  of  the 
mucous  membrane  there.  From  the  pus  in  cases  of  pyosalpinx 
they  have  been  cultivated  in  a  considerable  number  of  cases. 
According  to  the  results  of  various  observers  they  are  present 
in  one  out  of  four  or  five  cases  of  this  condition,  usually  un- 
as>ociated  with  other  organisms.  Further,  in  a  large  proj)ortion 
of  the  cases  in  which  the  gonococcus  has  not  been  found,  no 
organisms  of  any  kind  have  been  obtained  from  the  pus,  and 
in  these  cases  the  gonococci  may  have  been  once  present  and 
have  subsequently  died  out.  Lastly,  they  may  pass  to  the 
peritoneum  and  produce  peritonitis,  which  is  usually  of  a  local 
character.  It  is  chiefly  to  the  methods  of  culture  supplied  by 
\Yertheim  that  we  owe  our  extended  knowledge  of  such 
conditions. 

In  gonorrhoeal  conjunctivitis  the  mode  in  which  the  gonococci 
spread  through  the  epithelium  to  the  subjacent  connective 
tissue  is  closely  analogous  to  what  obtains  in  the  case  of  the 
urethra.  Their  relation  to  the  leucocytes  in  the  purulent 
secretion  is  also  the  same.  Microscopic  examination  of  the 
secretion  alone  in  acute  cases  often  gives  positive  evidence,  and 
I  mre  cultures  may  be  readily  obtained  on  blood-agar.  As  the 
condition  becomes  more  chronic  the  gonococci  are  less  numerous 
and  a  greater  proportion  of  other  organisms  may  be  present. 

Relations  to  Joint-Affections,  etc. — The  relations  of  the  gono- 


256  GONORRHCEA  AND  SOFT  SORE 

coccus  to  the  sequelae  of  gonorrhoea  form  a  subject  of  great 
interest  and  importance,  and  the  application  of  recent  methods 
of  examination  shows  that  the  organism  is  much  more  frequently 
present  in  such  conditions  than  the  earlier  results  indicated. 
The  following  statements  may  be  made  with  regard  to  them  : 
First,  in  a  large  number  of  cases  of  arthritis  following  gonorrhoea 
pure  cultures  of  the  gonococcus  may  be  obtained.  A  similar 
statement  applies  to  inflammation  of  the  sheaths  of  tendons 
following  gonorrhoea.  Secondly,  in  a  considerable  proportion  of 
cases  no  organisms  have  been  found.  It  is,  however,  possible 
that  in  many  of  these  the  gonococci  may  have  been  present 
in  the  synovial  membrane,  as  it  has  been  observed  that  they 
may  be  much  more  numerous  in  that  situation  than  in  the 
fluid.  Thirdly,  in  some  cases,  especially  in  those  associated 
with  extensive  suppuration,  occasionally  of  a  pya3mic  nature, 
various  pyogenic  cocci  have  been  found  to  be  present.  In  the 
instances  in  which  the  gonococcus  has  been  found  in  the  joints, 
the  fluid  present  has  usually  been  described  as  being  of  a 
whitish  yellow  tint,  somewhat  turbid,  and  containing  shreds 
of  fibrin-like  material,  sometimes  purulent  in  appearance.  In 
one  case  Bordoni-Uffreduzzi  cultivated  the  gonococcus  from  a 
joint  -  affection,  and  afterwards  produced  gonorrhoea  in  the 
human  subject  by  inoculating  with  the  cultures  obtained.  In 
another  case,  in  which  pleurisy  was  present  along  with  arthritis, 
the  gonococcus  was  cultivated  from  the  fluid  in  the  pleural 
cavity.  The  existence  of  a  gonorrhoeal  endocarditis  has  been 
established  by  recent  observations.  Cases  apparently  of  this 
nature  occurring  in  the  course  of  gonorrhoea  had  been  previously 
described,  but  the  complete  bacteriological  test  has  now  been 
satisfied  in  several  instances.  In  one  case  Lenhartz  produced 
gonorrhoea  in  the  human  subject  by  inoculation  with  the 
organisms  obtained  from  the  vegetations.  That  a  true 
gonorrhoeal  septicaemia  may  occur  has  also  been  established, 
cultures  of  the  gonococcus  having  been  obtained  from  the 
blood  during  life  on  more  than  one  occasion  (Thayer  and 
Blumer,  Thayer  and  Lazear,  Ahmann). 

Methods  of  Diagnosis. — For  microscopical  examination,  dried 
films  of  the  suspected  pus,  etc.,  may  be  stained  by  any  of  the 
simple  solutions  of  the  basic  aniline  stains.  We  prefer  methy- 
lene-  or  thionin-blue,  as  they  do  not  overstain,  and  the  films  do 
not  need  to  be  decolorised.  Staining  for  one  minute  is  sufficient. 
It  is  also  advisable  to  stain  by  Gram's  method,  and  it  is  a  good 
plan  to  put  at  one  margin  of  the  cover-glass  a  small  quantity  of 
culture  of  staphylococcus  aureus  if  available,  in  order  to  have 


SOFT  SOUK  257 

a  standard  by  which  to  be  certain  that  the  supposed  gonococci 
are  really  decolorised.  Regarding  the  value  of  microscopic 
examination  alone,  we  may  say  that  the  presence  of  a  large 
number  of  micrococci  in  a  urethral  discharge  having  the 
characters,  position,  and  staining  reactions  described  above, 
is  practically  conclusive  that  the  case  is  one  of  gonorrhea. 
There  is  no  other  condition  in  which  the  sum-total  of  the 
microscopical  characters  is  present.  We  consider  that  it  is 
sufficient  for  purposes  of  clinical  diagnosis,  and  therefore 
of  great  value ;  in  the  acute  stage  a  diagnosis  can  thus  be 
made  earlier  than  by  any  other  method.  The  mistake  of 
confusing  gonorrhoea  with  such  conditions  as  a  urethral  chancre 
with  urethritis,  will  also  be  avoided.  Even  in  chronic  cases 
the  typical  picture  is  often  well  maintained,  and  microscopic 
examination  alone  may  give  a  definite  positive  result.  When 
other  organisms  are  present,  and  especially  when  the  gonococci 
are  few  in  number,  it  is  difficult,  and  in  some  cases  impossible, 
to  give  a  definite  opinion,  as  a  few  gonococci  mixed  with  other 
organisms  cannot  be  recognised  with  certainty.  This  is  often 
the  condition  in  chronic  gonorrhoea  in  the  female.  Microscopic 
examination,  therefore,  though  often  giving  positive  results, 
will  sometimes  be  inconclusive.  As  regards  lesions  in  other 
parts  of  the  body,  microsocopic  examination  alone  is  quite 
insufficient ;  it  is  practically  impossible,  for  example,  to 
distinguish  by  this  means  the  gonococcus  from  the  diplococcus 
intracellularis  of  meningitis.  Cultures  alone  supply  the  test, 
and  the  points  above  detailed  are  to  be  attended  to. 

SOFT  SOKK. 

Tin;  bacillus  of  soft  sore  was  first  described  by  Ducrey  in 
1889,  who  found  it  in  the  purulent  discharge  from  the  ulcerated 
surface;  and  later,  in  1892,  Unna  described  its  appearance  and 
distribution  as  seen  in  sections  through  the  sores.  The  state- 
ments of  these  observers  regarding  the  presence  and  characters 
of  this  organism  have  been  fully  confirmed  by  other  observers. 

Microscopical  Characters. — The  organism  occurs  in  the  form 
of  minute  oval  rods  measuring  about  1*5  ft  in  length,  and  *5  ft 
in  thickness  (Fig.  75).  It  is  found  mixed  with  other  organisms 
in  the  purulent  discharge  from  the  surface,  and  is  chiefiy  arranged 
in  small  groups  or  in  short  chains.  When  studied  in  sections 
through  the  ulcer,  it  is  found  in  the  superficial  part  of  the  floor, 
but  more  deeply  situated  than  other  organisms,  and  may  be 
present  in  a  state  of  purity  amongst  the  leucocytic  infiltration. 


258 


GONORRHOEA  AND  SOFT  SORE 


In  this  position  it  is  usually  arranged  in  chains,  which  may  be 
of  considerable  length,  and  w^hich  are  often  seen  lying  in  parallel 
rows  between  the  cells.  The  bacilli  chiefly  occur  in  the  free 
condition,  but  occasionally  a  few  may  be  contained  within 
leucocytes. 

There  is  no  doubt  that  in  many  cases  the  organism  is  present 
in  the  buboes  in  a  state  of  purity ;  it  has  been  found  there  by 
microscopic  examination,  and  cultures  have  also  been  obtained 

from  this  source.  The 
negative  results  of  some 
observers  are  probably 
due  to  the  organism 
having  died  off.  On  the 
whole  the  evidence  goes 

§>*  w        fJJff       £F\     to  show  tliat  tlie  ordinary 
(flHllH^' KB      J     bubo  associated  with  soft 
*•  i^JjJpBJr     Kl  fm     sore  is  to  be  regarded  as 
^&  ^jlgiKp     ^     another   lesion    produced 

*^  *  by     Ducrey's       bacillus. 

Sometimes  the  ordinary 
pyogenic  organisms  be- 
come superadded. 

This  bacillus  takes  up 
the   basic    aniline    stains 
FIG.  75.— Film  preparation  of  pus  from  soft    fairly   readily,    but  loses 
chancre,  showing  Ducrey's  bacillus,  chiefly    t^      Pnlnnr    vprv    ranirllv 
arranged   in    pairs.     Stained  with   carbol-    l  _ve!7    iaPlcll> 

fuchsin  and  slightly  decolorised,     x  1500.      when  a  decolorising  agent 

is  applied.     Accordingly, 

in  film  preparations  when  dehydration  is  not  required,  it  can 
be  readily  stained  by  most  of  the  ordinary  combinations,  though 
Loffler's  or  Kiihne's  methylene-blue  solutions  are  preferable,  as 
they  do  not  overstain.  In  sections,  however,  great  care  must 
be  taken  in  the  process  of  dehydration,  and  the  aniline-oil 
method  (vide  p.  100)  should  be  used  for  this  purpose,  as  alcohol 
decolorises  the  organism  very  readily.  A  little  of  the  methylene- 
blue  or  other  stain  may  be  with  advantage  added  to  the  aniline- 
oil  used  for  dehydrating. 

Cultivation. — Although  for  a  long  period  of  time  attempts 
to  obtain  cultures  were  unsuccessful,  success  has  been  attained 
within  recent  years.  Bezancon,  Griffon,  and  Le  Sourd  obtained 
pure  cultures  in  four  cases,  the  medium  used  being  a  mixture  of 
rabbit's  blood  and  agar,  in  the  proportion  of  one  part  of  the 
former  to  two  of  the  latter.  The  blood  is  added  to  the  agar  in 
the  melted  condition  at  45°  C.,  and  the  tubes  are  then  sloped. 


SOFT  SORE 


259 


Davis  confirms  these  results,  and  finds  that  another  good  medium 
is  freshly-drawn  human  blood  distributed  in  small  tubes;  this 
method  is  specially  suitable,  as  the  blood  inhibits  the  growth 
of  various  extraneous  organisms.  On  the  solid  medium  (blood- 
agar)  the  growth  appears  in  the  form  of  small  round  globules, 
which  attain  their  complete  development  in  forty-eight  hours, 
having  then  a  diameter  of  1  to  2  mm. ;  the  colonies  do  not 
become  confluent.  Microscopic  examination  of  these  colonies, 
which  are  dissociated 
with  some  difficulty, 
shows  appearances  simi- 
lar to  those  observed 
when  the  organism  is 
in  the  tissues  (Fig.  76), 
but  occasionally  long 
undivided  filaments  are 
observed  which  Davis 
regards  as  degenerative 
forms.  Within  a  com- 
paratively short  period 
cultures  undergo  marked 
degenerative  changes, 
and  great  irregularities 
of  form  arid  shape  are  to 
be  found.  It  would  ap- 
pear that  a  comparatively 
large  amount  of  blood  is 

necessary  for  the  growth  of  this  organism,  and  even  sub-cultures 
on  the  ordinary  media,  including  blood -serum  media,  give 
negative  results.  Inoculation  of  the  ordinary  laboratory  animals 
is  not  attended  by  any  result,  but  it  has  been  found  that  some 
monkeys  are  susceptible,  small  ulcerations  being  produced  by 
superficial  inoculation,  and  in  these  the  organism  can  be  demon- 
strated. Tomasczewski  cultivated  the  organism  for  several 
generations,  and  reproduced  the  disease  by  inoculation  of  the 
human  subject.  The  causal  relationship  of  this  bacillus  must 
therefore  be  considered  as  completely  established,  and  the  con- 
ditions under  which  it  grows  show  it  to  be  a  strict  parasite — a 
fact  which  is  in  conformity  with  the  known  facts  as  to  the 
transmission  of  the  disease. 

1  We  are  indebted  to  Dr.  Davis  for  the  use  of  Figs.  75  and  76, 


j&.-'-v 

^x$ 


FIG.  76. — Ducrey's  bacillus  from  a  24-hour 
culture  in  blood-bouillon,      x  1500.1 


CHAPTER   X. 

TUBERCULOSIS. 

THE  cause  of  tubercle  was  proved  by  Koch  in  1882  to  be  the 
organism  now  universally  known  as  the  tubercle  bacillus. 
Probably  no  other  single  discovery  has  had  a  more  important 
effect  on  medical  science  and  pathology  than  this.  It  has  not 
only  shown  what  is  the  real  cause  of  the  disease,  but  has  also 
supplied  infallible  methods  for  determining  what  are  tubercular 
lesions  and  what  are  not,  and  has  also  given  the  means  of 
studying  the  modes  and  paths  of  infection.  A  definite  answer 
has  in  this  way  been  supplied  to  many  questions  which  were 
previously  the  subject  of  endless  discussion. 

Historical. — By  the  work  of  Armarmi  and  of  Cohnheim  and  Salomonsen 
(1870-80)  it  had  been  demonstrated  that  tubercle  was  an  infective  disease. 
The  latter  observers  found  on  inoculation  of  the  anterior  chamber  of  the 
eye  of  rabbits  with  tubercular  material,  that  in  many  cases  the  results  of 
irritation  soon  disappeared,  but  that  after  a  period  of  incubation,  usually 
about  twenty-five  days,  small  tubercular  nodules  appeared  in  the  iris  ; 
afterwards  the  disease  gradually  spread,  leading  to  a  tubercular  disorgan- 
isation of  the  globe  of  the  eye.  Later  still,  the  lymphatic  glands  became 
involved,  and  finally  the  animal  died  of  acute  tuberculosis.  The  question 
remained  as  to  the  nature  of  the  virus,  the  specific  character  of  which 
was  thus  established,  and  this  question  was  answered  by  the  work  of 
Koch. 

The  announcement  of  the  discovery  of  the  tubercle  bacillus  was  made 
by  Koch  in  March  1882,  and  a  full  account  of  his  researches  appeared  in 
1884  (Mirth,  a.  d.  K.  Gfsndhtsamte.,  Berlin).  Koch's  work  on  this  subject 
will  remain  as  a  classical  masterpiece  of  bacteriological  research,  both  on 
account  of  the  great  difficulties  which  he  successfully  overcame  and  the 
completeness  with  which  he  demonstrated  the  relations  of  the  organism 
to  the  disease.  The  two  chief  difficulties  Avere,  first,  the  demonstration 
of  the  bacilli  in  the  tissues,  and,  secondly,  the  cultivation  of  the  organism 
outside  the  body.  For,  with  regard  to  the  first,  the  tubercle  bacillus 
cannot  be  demonstrated  by  a  simple  watery  solution  of  a  basic  aniline 
dye,  and  it  was  only  after  prolonged  staining  for  twenty-four  hours,  with 
a  solution  of  methylene-blue  with  caustic  potash  added,  that  he  was 
able  to  reveal  the  presence  of  the  organism.  Then,  in  the  second  place, 
all  attempts  to  cultivate  it  on  the  ordinary  media  failed,  and  he  only 

260 


TUBERCULOSIS  IN  ANIMALS  261 

succeeded  in  obtaining  growth  on  solidified  blood  serum,  the  method  of 
preparing  which  he  himself  devised,  inoculations  being  made  on  this 
medium  from  the  organs  of  animals  artificially  rendered  tubercular. 
The  fact  that  growth  did  not  appear  till  the  tenth  day  at  the  earliest, 
might  easily  have  led  to  the  hasty  conclusion  that  no  growth  took  place. 
All  difficulties  were,  however,  successfully  overcome.  He  cultivated  the 
organism  by  the  above  method  from  a  great  variety  of  sources,  and  by 
a  large  series  of  inoculation  experiments  on  various  animals,  performed 
by  different  methods,  he  conclusively  proved  that  bacilli  from  these 
different  sources  produced  the  same  tubercular  lesions  and  were  really  of 
the  same  species.  His  work  was  the  means  of  showing  conclusively  that 
such  conditions  as  lupus,  "white  swelling"  of  joints,  scrofulous  disease 
of  glands,  etc.,  are  really  tubercular  in  nature. 

Tuberculosis  in  Animals. — Tuberculosis  is  not  only  the  most 
widely  spread  of  all  diseases  affecting  the  human  subject,  and 
produces  a  mortality  greater  than  any  other,  but  there  is  probably 
no  other  disease  which  affects  the  domestic  animals  so  widely. 
We  need  not  here  describe  in  detail  the  various  tubercular  lesions 
in  the  human  subject,  but  some  facts  regarding  the  disease  in 
the  lower  animals  may  be  given,  as  this  subject  is  of  great 
importance  in  relation  to  the  infection  of  the  human  subject. 

Amongst  the  domestic  animals  the  disease  is  commonest  in  cattle 
(bovine  tuberculosis),  in  which  animals  the  lesions  are  very  various,  both 
in  character  and  distribution.  In  most  cases  the  lungs  are  affected,  and 
contain  numerous  rounded  nodules,  many  being  of  considerable  size  ; 
these  may  be  softened  in  •  the  centre,  but  are  usually  of  pretty  firm 
consistence  and  may  be  calcified.  There  may  be  in  addition  caseous 
pneumonia,  and  also  small  tubercular  granulations.  Along  with  these 
changes  in  the  lungs,  the  pleurae  are  also  often  affected,  and  show 
numerous  nodules,  some  of  which  may  be  of  large  size,  firm  and  pedun- 
culated,  the  condition  being  known  in  Germany  as  Perlsucht,  in  France 
as  pommeliere.  Lesions  similar  to  the  last  may  be  chiefly  confined  to 
the  peritoneum  and  pleune.  In  other  cases,  again,  the  abdominal  organs 
are  principally  involved.  The  udder  becomes  affected  in  a  certain  pro- 
portion of  cases  of  tuberculosis  in  cows — in  3  per  cent,  according  to  Bang 
— but  primary  affection  of  this  gland  is  very  rare.  Tuberculosis  is  also 
a  comparatively  common  disease  in  pigs,  in  which  animals  it  in  many 
cases  affects  the  abdominal  organs,  in  other  cases  produces  a  sort  of 
osseous  pneumonia,  and  sometimes  is  met  with  as  a  chronic  disease  of 
the  lymphatic  glands,  the  so-called  "scrofula"  of  pigs.  Tubercular 
lesions  in  the  muscles  are  less  rare  in  pigs  than  in  most  other  animals. 
In  the  horse  the  abdominal  organs  are  usually  the  primary  seat  of  the 
disease,  the  spleen  being  often  enormously  enlarged  and  crowded  with 
nodules  of  various  shapes  and  sizes  ;  sometimes,  however,  the  primary 
lesions  are  pulmonary.  In  sheep  and  goats  tuberculosis  is  of  rare 
occurrence,  especially  in  the  former  animals.  It  may  occur  spontaneously 
in  dogs,  cats,  and  in  the  large  carnivora.  It  is  also  sometimes  met  with 
in  monkeys  in  confinement,  and  leads  to  a  very  rapid  and  widespread 
affection  in  these  animals,  the  nodules  having  a  special  tendency  to 
soften  and  break  down  into  a  pus-like  fluid. 


262  TUBERCULOSIS 

Tuberculosis  in  fowls  (avian  tuberculosis)  is  a  common  and  very 
infectious  disease,  nearly  all  the  birds  in  a  poultry-yard  being  sometimes 
affected. 

From  these  statements  it  will  be  seen  that  the  disease  in 
animals  presents  great  variations  in  character,  and  may  differ  in 
many  respects  from  that  met  with  in  the  human  subject.  The 
relation  of  the  different  forms  of  tuberculosis  is  discussed  below. 
Tubercle  Bacillus— Microscopical  Characters. — Tubercle 
bacilli  are  minute  rods  which  usually  measure  2 -5  to  3 -5  //,  in 
length,  and  '3  /x  in  thickness,  i.e.  in  proportion  to  their  length 
they  are  comparatively  thin  organisms  (Figs.  77  and  78).  Some- 
times, however,  longer 
forms,  up  to  5  /x  or  more 
in  length,  are  met  with, 
both  in  cultures  and  in 
the  tissues.  They  are 
straight  or  slightly  curved, 
and  are  of  uniform  thick- 
ness, or  may  show  slight 
swelling  at  their  extremi- 
ties;  When  stained  they 
appear  uniformly  colour- 
ed, or  may  present  small 
uncoloured  spots  along 
their  course,  with  darkly 
stained  parts  between.  In 

such  a  minute   organism 
FIG.  It. — Tubercle  bacilli,  from  a  pure         ...  -,       J-&,      u 

culture  on  glycerin  agar.  ^    1S     extremely    difficult 

Stained  with  carbol-fuchsin.     x  1000.          to    determine    the    exact 

nature    of   the  unstained 

points.  Accordingly,  we  find  that  some  observers  consider 
these  to  be  spores,  while  others  find  that  it  is  impossible  to 
stain  them  by  any  means  whatever,  and  consider  that  they 
are  really  of  the  nature  of  vacuoles.  Against  their  being 
spores  is  also  the  fact  that  many  occur  in  one  bacillus.  Others 
again  hold  that  some  of  the  condensed  and  highly  stained 
particles  are  spores.  It  is  impossible  to  speak  definitely  on  the 
question  at  present.  We  can  only  say  that  the  younger  bacilli 
stain  uniformly,  and  that  in  the  older  forms  inequality  in  stain- 
ing is  met  with ;  this  latter  condition  is,  however,  not  found  to 
be  associated  with  greater  powers  of  resistance. 

The  bacilli  in  the  tissues  occur  scattered  irregularly  or  in 
little  masses.  They  are  usually  single,  or  two  are  attached  end 
to  end  and  often  form  in  such  a  case  an  obtuse  angle.  True 


THE  TUBERCLE  BACILLUS  263 

chains~are  not  formed,  but  occasionally  short  filaments  are  met 
with.  In  cultures  the  bacilli  form  masses  in  which  the  rods  are 
closely  applied  to  one  another  and  arranged  in  a  more  or  less 
parallel  manner.  Tubercle  bacilli  are  quite  devoid  of  motility. 
Aberrant  Forms. — Though  such  are  the  characters  of  the 
organism  as  usually  met  with,  other  appearances  are  sometimes 
found.  In  old  cultures,  for  example,  very  much  larger  elements 


FIG.  78. — Tubercle  bacilli  iu  phthisical  sputum  ;  they  are  longer  than 

is  often  the  case.     See  also  Plate  II.,  Fig.  7. 

Film  preparation,  stained  with  carbol-fuchsin  and  inethylene-blue. 
xlOOO. 

may  occur.  These  may  be  in  the  form  of  long  filaments,  some- 
times swollen  or  clubbed  at  their  extremities,  may  be  irregularly 
beaded,  and  may  even  show  the  appearance  of  branching.  Such 
forms  have  been  studied  by  Metchnikoff,  Maffucci,  Klein,  and 
others.  Their  significance  has  been  variously  interpreted,  for 
while  some  look  upon  them  as  degenerated  or  involution  forms, 
others  regard  them  as  indicating  a  special  phase  in  the  life 
history  of  the  organism,  allying  it  with  the  higher  bacteria. 
Recent  observations,  however,  go  to  establish  the  latter  view, 
and  this  is  now  generally  accepted  by  authorities.  It  has  also 


264  TUBERCULOSIS 

been  found  that  under  certain  circumstances  tubercle  bacilli  in 
the  tissues  produce  a  radiating  structure  closely  similar  to  that 
of  the  actinomyces.  This  was  found  by  Babes  and  also  by 
Lubarsch  to  be  the  case  when  the  bacilli  were  injected  under 
the  dura  mater  and  directly  into  certain  solid  organs,  such  as 
the  kidneys  in  the  rabbit.  Club-like  structures  may  be  present 
at  the  periphery ;  these  are  usually  not  acid-fast,  but  they  retain 
the  stain  in  the  Weigert-Gram  method.  Similar  results  obtained 
with  other  acid-fast  bacilli  will  be  mentioned  below,  and  these 
organisms  would  appear  to  form  a  group  closely  allied  to  the 
streptothricese,  the  bacillary  parasitic  form  being  one  stage  of 
the  life  history  of  the  organism.  This  group  is  often  spoken  of 
as  the  mycobacteria. 

Staining  Reactions.  —  The  tubercle  bacillus  takes  up  the 
ordinary  stains  very  slowly  and  faintly,  and  for  successful  stain- 
ing one  of  the  most  powerful  solutions  ought  to  be  employed,  e.g. 
gentian-violet  or  fuchsin,  along  with  aniline-oil  water  or  solution 
of  carbolic  acid.  Further,  such  staining  solutions  require  to  be 
applied  for  a  long  time,  or  the  staining  must  be  accelerated  by 
heat,  the  solution  being  warmed  till  steam  arises  and  the 
specimen  allowed  to  remain  in  the  hot  stain  for  two  or  three 
minutes.  One  of  the  best  and  most  convenient  methods  is  the 
Ziehl-Neelsen  method  (see  p.  108).  -The  bacilli  present  this 
further  peculiarity,  however,  that  after  staining  has  taken  place 
they  resist  decolorising  by  solutions  which  readily  remove  the 
colour  from  the  tissues  and  from  other  organisms  which  may  be 
present.  Such  decolorising  agents  are  sulphuric  or  nitric  acid 
in  20  per  cent,  solution.  Preparations  can  thus  be  obtained  in 
which  the  tubercle  bacilli  alone  are  coloured  by  the  stain  first 
used,  and  the  tissues  can  then  be  coloured  by  a  contrast  stain. 
Within  recent  years  certain  other  bacilli  have  been  discovered 
which  present  the  same  staining  reactions  as  tubercle  bacilli ; 
they  are  therefore  called  "  acid-fast "  (vide  infra).  The  spores 
of  many  bacilli  become  decolorised  more  readily  than  tubercle 
bacilli,  though  some  retain  the  colour  with  equal  tenacity. 

Bullocli  and  Macleod,  by  treating  tubercle  bacilli  with  hot  alcohol 
and  ether,  extracted  a  wax  which  gave  the  characteristic  staining 
reactions  of  the  bacilli  themselves.  The  remains  of  the  bacilli,  further, 
when  extracted  with  caustic  potash,  yielded  a  body  which  was  probably 
a  chitin,  and  which  was  acid-fast  when  stained  for  twenty-four  hours 
with  carbol-fuchsin. 

It  had  long  been  recognised  that  it  might  not  be  possible  to 
detect  by  microscopic  methods  tubercle  bacilli  in  old  tubercular 


CULTIVATION  OF  TUBERCLE  BACILLUS       265 

lesions,  and  yet  the  material  from  such  was  virulent  on 
inoculation.  This  was  supposed  to  be  due  either  to  the 
paucity  of  the  bacilli  or  possibly  to  the  presence  of  spores. 
Recently  observations  have  been  brought  forward  by  Much 
which  may  throw  important  light  on  this  subject.  Briefly  put, 
his  conclusions  are  that  the  tubercle  virus  exists  in  three  forms 
— (a)  the  ordinary  bacillary  form  stainable  by  the  Ziehl  method  ; 
(6)  a  fine  bacillary  form  which  is  not  acid-fast,  often  showing 
granules  in  its  interior ;  and  (c)  free  granules  which  also  fail  to 
stain  with  the  Ziehl  method.  The  two  last  forms  can  be  stained 
by  Gram's  method  when  the  stain  is  applied  for  a  long  time. 
Much  gives  three  modifications  of  Gram's  method,  the  following 
being  one  which  has  been  found  by  others  to  be  specially 
suitable : — 

Methyl  violet  B.  N.  10  c.  c.  of  a  saturated  alcoholic  solution  in  100  c.c.  of 
a  2  per  cent,  watery  solution  of  carbolic  acid  ;  stain  by  boiling  over  the 
name  for  a  few  minutes  or  at  37°  C.  for  24-48  hours,  then  treat  with  Lugoe's 
iodine  for  1-5  minutes,  5  per  cent,  hydrochloric  acid  for  one  minute,  3 
per  cent,  hydrochloric  acid  10  seconds,  and  complete  the  decolorisation 
with  ;i  mixture  of  acetone  and  alcohol  in  equal  parts. 

Much  claims  that  by  such  a  method  bacilli  and  granules  can 
be  found  in  tubercular  lesions  when  the  Ziehl  method  gives 
a  negative  result.  He  also  found  that,  w^ien  bacilli  from  a 
culture  were  added  to  sterilised  milk  and  incubated,  the  acid-fast 
forms  disappeared  whilst  those  stainable  with  Gram's  method 
remained;  and  that  when  this  had  occurred  the  milk  when 
injected  into  an  animal  produced  tuberculosis  in  which  acid-fast 
bacilli  were  demonstrable.  His  statements  have  received  con- 
firmation by  other  observers,  e.g.  Wirths  and  Treuholtz,  but  as 
yet  it  is  not  possible  to  give  a  definite  pronouncement  on  the 
whole  subject.  If  the  bacillus  can  pass  into  a  form  not  demon- 
strable by  the  method  usually  employed  but  still  virulent,  we 
have  manifestly  to  deal  with  a  fact  of  the  highest  importance. 
There  seems  to  be  no  doubt  that  in  certain  conditions  more 
tubercle  bacilli  can  be  demonstrated  in  the  tissues  by  Much's 
method  than  by  the  ordinary  carbol-f  uchsin  method. 

Cultivation. — The  medium  first  used  by  Koch  was  inspissated 
blood  serum  (vide  p.  40).  If  inoculations  are  made  on  this 
medium  with  tubercular  material  free  from  other  organisms, 
there  appear  in  from  ten  to  fourteen  days  minute  points  of  growth 
of  dull  whitish  colour,  rather  irregular,  and  slightly  raised  above 
the  surface  (it  is  advisable  to  plant  on  the  medium  an  actual 
piece  of  the  tubercular  tissue  and  to  fix  it  in  a  wound  of  the 


266 


TUBERCULOSIS 


surface  of  the  serum).  Koch  compared  the  appearance  of  these 
to  that  of  small  dry  scales.  In  such  cultures  the  growths  usually 
reach  only  a  comparatively  small  size  and  remain  separate,  be- 
coming confluent  only  when  many  occur  close  together.  In  sub- 
cultures, however,  growth  is  more  luxuriant  and  may  come  to 
form  a  dull  wrinkled  film  of  whitish  colour,  which  may  cover 

the  greater  part  of  the 
surface  of  the  serum  and 
at  the  bottom  of  the  tube 
may  grow  over  the  sur- 
face of  the  condensation 
water  on  to  the  glass 
(Fig.  79,  A).  The  growth 
is  always  of  a  dull  ap- 
pearance, and  has  a  con- 
siderable degree  of  con- 
sistence, so  that  it  is 
difficult  to  dissociate  a 
portion  thoroughly  in  a 
drop  of  water.  In  older 
cultures  the  growth  may 
acquire  a  slightly  brown- 
ish or  buff  colour.  When 
the  small  colonies  are 
examined  under  a  low 
power  of  the  microscope, 
they  are  seen  to  be  ex- 
tending at  the  periphery 
in  the  form  of  wavy  or 
sinuous  streaks  which 
radiate  outward,  and 
which  have  been  com- 
pared to  the  flourishes  of 
a  pen.  The  central  part 
shows  similar  markings 
closely  interwoven.  These 
streaks  are  composed  of  masses  of  the  bacilli  arranged  in  a  more 
or  less  parallel  manner. 

On  glycerin  agar,  which  was  first  introduced  by  Nocard  and 
Roux  as  a  medium  for  the  culture  of  the  tubercle  bacillus, 
growth  takes  place  in  sub-cultures  at  an  earlier  date  and  pro- 
gresses more  rapidly  than  on  serum,  but  this  medium  is  not 
suitable  for  obtaining  cultures  from  the  tissues,  inoculations 
with  tubercular  material  usually  yielding  a  negative  result. 


FIG.  79. — Cultures  of  tubercule  bacilli  on 
glycerin  agar. 

A  and  B.     Mammalian  tubercle  bacilli ;  A  is  an 

old  culture,  B  one  of  a  few  weeks'  growth. 

C.    Avian  tubercle  bacilli.    The  growth  is  whiter 

and  smoother  on  the  surface  than  the  others. 


CULTIVATION  OF  TUBERCLE  BACILLUS       267 

The  growth  has  practically  the  same  characters  as  on  serum,  but 
is  more  luxuriant.  In  glycerin  broth,  especially  when  the  layer 
is  not  deep,  tubercle  bacilli  grow  readily  in  the  form  of  little 
white  masses,  which  fall  to  the  bottom  and  form  a  powdery  layer. 
If,  however,  the  growth  be  started  on  the  surface,  it  spreads 
superficially  as  a  dull  whitish  wrinkled  pellicle  which  may  reach 
the  walls  of  the  flask ;  this  mode  of  growth  is  specially  suitable 
for  the  production  of  tuberculin  (vide  infra).  The  culture  has 
a  peculiar  fruity  and  not  unpleasant  odour.  On  ordinary  agar 
and  on  gelatin  media  no  growth  takes  place.  The  use  of  animal 
tissues  in  glycerine  bouillon  as  a  medium  for  the  growth  of  the 
tubercle  bacillus  has  been  recently  introduced  by  Frugoni,  and 
is  one  which  gives  excellent  results.  He  recommends  that  small 
wedges  of  rabbit's  lung  should  be  sterilised  in  the  autoclave,  and 
placed  in  tubes  of  glycerine  bouillon  in  such  a  way  that  their 
surface  is  kept  moist  by  the  medium,  without  the  fragments 
being  submerged.  The  growth  is  probably  more  rapid  and 
luxuriant  than  in  any  other  method. 

Use  of  Egg  Media. — Within  recent  years  media  containing 
either  the  yolk  or  both  the  yolk  and  the  white  of  egg  have  been 
used  for  the  culture  of  the  tubercle  bacillus  by  Dorset  and  others. 
The  following  is  Dorset's  method :  The  contents  of  four  eggs 
are  well  beat,  25  c.c.  of  water  are  added  and  thoroughly  mixed, 
the  mixture  being  passed  through  muslin  to  remove  air  bells. 
The  fluid  is  then  filled  into  tubes,  and  these  are  heated  for  four 
hours  in  the  sloped  position  at  70°  C.  Before  the  inoculation  of 
a  tube,  two  drops  of  sterilised  water  are  placed  on  the  surface. 
The  inoculation  material  is  well  rubbed  over  the  surface  of  the 
medium,  the  tubes  are  sealed  with  a  few  drops  of  paraffin  on  the 
top  of  the  plug  and  are  incubated  in  the  sloped  position. 
Vigorous  growth  takes  place  on  such  media,  having,  generally 
speaking,  the  naked  eye  characters  seen  in  blood  serum  cultures. 

It  was  at  one  time  believed  that  the  tubercle  bacillus  would  only  grow 
on  media  containing  animal  fluids,  but  of  late  years  it  has  been  found 
that  growth  takes  place  also  on  a  purely  vegetable  medium,  as  was  first 
shown  by  Pawlowsky  in  the  case  of  potatoes.  Sander  found  that  the 
bacillus  grew  readily  on  potato,  carrot,  macaroni,  and  on  infusion  of 
these  substances,  especially  when  glycerin  was  added.  He  also  found 
that  cultures  from  tubercular  lesions  could  be  obtained  on  glycerin  potato 
(p.  46). 

The  optimum  tcmi>craturc  tor  growth  is  37°  to  38°  C. 
Growth  ceases  about  42°  and  usually  below  28°,  but  on  long- 
continued  cultivation  outside  the  body  and  in  special  circum- 
stances, growth  may  take  place  at  a  lower  temperature,  e.g. 


268  TUBERCULOSIS 

Sander  found  that  growth  took  place  in  glycerin-potato  broth 
even  at  22°  to  23°  C. 

Powers  of  Resistance. — Tubercle  bacilli  have  considerable 
powers  of  resistance  to  external  influences,  and  can  retain  their 
vitality  for  a  long  time  outside  the  body  in  various  conditions ; 
in  fact,  in  this  respect  they  may  be  said  to  occupy  an  inter- 
mediate position  between  spores  and  spore-free  bacilli.  Dried 
phthisical  sputum  has  been  found  to  contain  still  virulent  bacilli 
(or  their  spores)  after  two  months,  and  similar  results  are  obtained 
when  the  bacilli  are  kept  in  distilled  water  for  several  weeks. 
So  also  they  resist  for  a  long  time  the  action  of  putrefaction, 
which  is  rapidly  fatal  to  many  pathogenic  organisms.  Sputum 
has  been  found  to  contain  living  tubercle  bacilli  even  after  being 
allowed  to  putrefy  for  several  weeks  (Fraenkel,  Baumgarten),  and 
the  bacilli  have  been  found  to  be  alive  in  tubercular  organs  which 
have  been  buried  in  the  ground  for  a  similar  period.  They  are 
not  killed  by  being  exposed  to  the  action  of  the  gastric  juice  for 
six  hours,  or  to  a  temperature  of  -  3°  C.  for  three  hours,  even 
when  this  is  repeated  several  times.  It  has  been  found  that 
when  completely  dried  they  can  resist  a  temperature  of  100°  C. 
for  an  hour,  but,  on  the  other  hand,  exposure  in  the  moist 
condition  to  70°  C.  for  the  same  time  is  usually  fatal.  It  may 
be  stated  that  raising  the  temperature  to  100°  C.  kills  the  bacilli 
in  fluids  and  in  tissues,  but  in  the  case  of  large  masses  of  tissue 
care  must  be  taken  that  this  temperature  is  reached  throughout. 
They  are  killed  in  less  than  a  minute  by  exposure  to  5  per  cent, 
carbolic  acid,  and  both  Koch  and  Straus  found  that  they  are 
rapidly  killed  by  being  exposed  to  the  action  of  direct  sunlight. 

Action  on  the  Tissues. — The  local  lesion  produced  by  the 
tubercle  bacillus  is  the  well-known  tubercle  nodule,  the 
structure  of  which  varies  in  different  situations  and  according  to 
the  intensity  of  the  action  of  the  bacilli.  After  the  bacilli  gain 
entrance  to  a  connective  tissue  such  as  that  of  the  iris,  their 
first  action  appears  to  be  on  the  connective-tissue  cells,  which 
become  somewhat  swollen  and  undergo  mitotic  division,  the 
resulting  cells  being  distinguishable  by  their  large  size  and  pale 
nuclei — the  so-called  epithelioid  cells.  These  prolif  erative  changes 
may  be  well  seen  on  the  fifth  day  after  inoculation  or  even 
earlier.  A  small  focus  of  proliferated  cells  is  thus  formed  in  the 
neighbourhood  of  the  bacilli,  and  about  the  same  time  numbers 
of  leucocytes — chiefly  lymphocytes — begin  to  appear  at  the 
periphery  and  gradually  become  more  numerous.  Soon,  however, 
the  action  of  the  bacilli  as  cell-poisons  comes  into  prominence. 
The  epithelioid  cells  become  swollen  and  somewhat  hyaline,  their 


ACTION  ON  THE  TISSUES  269 

outlines  become  indistinct,  whilst  their  nucleus  stains  faintly, 
and  ultimately  loses  the  power  of  staining.  The  cells  in  the 
centre,  thus  altered,  gradually  become  fused  into  a  homogeneous 
substance,  and  this  afterwards  becomes  somewhat  granular  in 
appearance.  If  the  central  necrosis  does  not  take  place  quickly, 
then  giant-cell  formation  may  occur  in  the  centre  of  the  follicle, 
this  constituting  one  of  the  characteristic  features  of  the  tuber- 
cular lesion ;  or  after  the  occurrence  of  caseation  giant-cells  may 
be  formed  in  the  cellular  tissue  around.  The  centre  of  a  giant- 
cell  often  shows  signs  of  degeneration,  such  as  hyaline  change 
and  vacuolation,  or  it  may  be  more  granular  than  the  rest  of 
the  cell.  The  exact  mode  of  formation  of  a  tubercle  follicle 
varies,  however,  in  different  tissues. 

Though  there  has  been  a  considerable  amount  of  discussion 
as  to  the  mode  of  origin  of  the  giant-cells,  we  think  there  can 
be  little  doubt  that  in  most  cases  they  result  from  enlargement 
of  single  epithelioid  cells,  the  nucleus  of  which  undergoes  pro- 
liferation without  the  protoplasm  dividing.  These  epithelioid 
cells  may  sometimes  be  the  lining  cells  of  capillaries.  Some 
consider  that  the  giant-cells  result  from  a  fusion  of  the  epithelioid 
cells ;  but,  though  there  are  occasionally  appearances  which 
indicate  such  a  mode  of  formation,  it  cannot  be  regarded  as  of 
common  occurrence.  In  some  cases  of  acute  tuberculosis,  when 
the  bacilli  become  lodged  in  a  capillary,  the  endothelial  cells  of 
its  wall  may  proliferate,  and  thus  a  ring  of  nuclei  may  be  seen 
round  a  small  central  thrombus.  Such  an  occurrence  gives  rise 
to  an  appearance  closely  resembling  a  typical  giant-cell. 
There  can  be  no  doubt  that  the  cell  necrosis  and  subsequent 
caseation  depend  upon  the  products  of  the  bacilli,  and  are  not 
due  to  the  fact  that  the  tubercle  nodule  is  non-vascular.  This 
non-vascularity  itself  is  to  be  explained  by  the  circumstance 
that  young  capillaries  cannot  grow  into  a  part  where  tubercle 
bacilli  are  active,  and  that  the  already  existing  capillaries  become 
thrombosed,  owing  to  the  action  of  the  bacillary  products  on 
their  walls,  and  ultimately  disappear.  At  the  periphery  of 
tubercular  lesions  there  may  be  considerable  vascularity  and  new 
formation  of  capillaries. 

The  general  symptoms  of  tuberculosis — pyrexia,  perspiration, 
wasting,  etc.,  are  to  be  ascribed  to  the  absorption  and  distribution 
throughout  the  system  of  the  toxic  products  of  the  bacilli ;  in 
the  case  of  phthisical  cavities  and  like  conditions  where  other 
bacteria  are  present,  the  toxins  of  the  latter  also  play  an  im- 
portant part.  The  occurrence  of  waxy  change  in  the  organs  is 
believed  by  some  to  be  chiefly  due  to  the  products  of  other, 


270 


TUBERCULOSIS 


especially  pyogenic,  organisms,  secondarily  present  in  the  tuber- 
cular lesions.  This  matter,  however,  requires  further  elucidation. 
Presence  and  Distribution-  of  the  Bacilli. — A  few  facts  may 
be  stated  regarding  the  presence  of  bacilli,  and  the  numbers  in 
which  they  are  likely  to  be  found  in  tubercular  lesions. 
They  are  usually  very  few  in  number  in  chronic  lesions, 
whether  these  are  tubercle  nodules  with  much  connective  tissue 


FIG.  80. — Tubercle  bacilli  in  section  of  human  lung  in  acute  phthisis. 
The  bacilli  are  seen  lying  singly,  and  also  in  large  masses  to  left  of 
field.  The  pale  background  is  formed  by  caseous  material. 

Stained  with  carbol-fuchsiu  and  Bismarck-brown,      x  1000. 


formation  or  old  caseous  collections.  In  caseous  material  one 
can  sometimes  see  a  few  bacilli  faintly  stained,  along  with  very 
minute  unequally  stained  granular  points,  some  of  which  may 
possibly  be  spores  of  the  bacilli.  Whether  they  are  spores  or 
not,  the  important  fact  has  been  established,  that  tubercular 
material  in  which  no  bacilli  can  be  found  microscopically,  may 
be  proved,  on  experimental  inoculation  into -animals,  to  be  still 
virulent.  In  such  cases  the  bacilli  may  be  present  in  numbers  so 
small  as  to  escape  observation,  or  it  may  be  that  their  spores  only 


ACTION  ON  THE  TISSUES 


271 


*ire  present.  In  subacute  lesions,  with  well-formed  tubercle 
follicles  and  little  caseation,  the  bacilli  are  generally  scanty. 
They  are  most  numerous  in  acute  lesions,  especially  where 
caseation  is  rapidly  spreading,  for  example,  in  such  conditions  as 
case.  HI*  catarrlial  pneumonia  (Fig.  80),  acute  tuberculosis  of  the 
spleen  in  children,  which  is  often  attended  with  a  good  deal  of 
rapid  casemis  change,  etc.  ;  in  such  conditions  they  often  form 


*"• 


Fi<;.  81.—  Tubercle  bacilli  in  giant-cells,  showing  the  radiate 
arrangement  at  the  periphery  of  the  cells.  Section  of  tubercular 
udder  of  cow. 

Stained  with  carbol-fuchsin  and  Rismarrk-brown.      x  1000. 

large  masses  which  are  easily  seen  under  a  low  power  of  the 
microscope.  In  acute  miliary  tuberculosis  a  few  bacilli  can 
generally  be  found  in  the  centre  of  the  follicles ;  but  here  they 
are  often  much  more  scanty  than  one  would  expect.  The 
tubercle  bacillus  is  one  which  not  only  has  comparatively  slow 
growth,  but  retains  its  form  and  staining  power  for  a  much 
longer  period  than  most  organisms.  As  a  rule  the  bacilli  are 
-extra-cellular  in  jK)sition.  Occasionally  they  occur  within  the 
iriant -cells,  in  which  they  may  lie  arranged  in  a  somewhat  radiate 


272 


TUBERCULOSIS 


manner  at  the    periphery,  occasionally  also  in  epithelioid  cells 
and  in  leucocytes. 

The  above  statements,  however,  apply  only  to  tuberculosis 
in  the  human  subject,  and  even  in  this  case  there  are  exceptions. 
In  the  ox,  on  the  other  hand,  the  presence  of  tubercle  bacilli 
within  giant-cells  is  a  very  common  occurrence  ;  and  it  is 
also  common  to  find  them  in  considerable  numbers  scattered 
irregularly  throughout  the  cellular  connective  tissue  of  the  lesions,. 
even  when  there  is  little  or  no  caseation  present  (Fig.  81). 

In  tuberculosis  in  the  horse  and  in  avian  tuberculosis  the 
numbers  of  bacilli  may  be  enormous,  even  in  lesions  which  are 

not  specially  acute  ;  and 
considerable  variation 
both  in  their  number  and 

4N91  ^          in  their  site  is  met  with 

in  tuberculosis  of  other 
animals. 

In  discharges  from 
tubercular  lesions  which 
are  breaking  down,  tu- 
bercle bacilli  are  usually 
to  be  found.  In  the 
sputum  of  phthisical 
patients  their  presence 
can  be  demonstrated  al- 
most invariably  at  some 
period,  and  sometimes 

FIG.  82.—  Tubercle  bacilli  in  urine  ;  showing     their    numbers    are    very 

one  of  the  characteristic  clumps,  in  which     j  (for  method  of  stain_ 

tney  oiten  occur.  .     •  r\**\      o  i 

Stained  with  carbol-fnchsin  and  methylene-     ing,  see  p.  10/).     Several 

blue.     xlOOO. 


examnatons    may, 
ever,  require  to  be  made  ; 

this  should  always  be  done  before  any  conclusion  as  to  the  non- 
tubercular  nature  of  a  case  is  come  to.  In  cases  of  genito-urinary 
tuberculosis  they  are  usually  present  in  the  urine  ;  but  as  they 
are  much  diluted  it  is  difficult  to  find  them  unless  a  deposit  is 
obtained  by  means  of  the  centrifuge.  This  deposit  is  examined 
in  the  same  way  as  the  sputum.  The  bacilli  often  occur  in  little 
clumps,  as  shown  in  Fig.  82.  In  tubercular  ulceration  of  the 
intestine  their  presence  in  the  faeces  may  be  demonstrated,  as 
was  first  shown  by  Koch  ;  but  in  this  case  their  discovery  is 
usually  of  little  importance,  as  the  intestinal  lesions,  as  a  rule, 
occur  only  in  advanced  stages  when  diagnosis  is  no  longer  a 
matter  of  doubt. 


EXPERIMENTAL  INOCULATION  273 

Experimental  Inoculation. — Tuberculosis  can  be  artificially 
produced  in  animals  by  infection  in  a  great  many  different  ways 
— by  injection  of  the  bacilli  into  the  subcutaneous  tissue,  into 
the  peritoneum,  into  the  anterior  chamber  of  the  eye,  into  the 
veins;  by  feeding  the  animals  with  the  bacilli;  and,  lastly,  by 
making  them  inhale  the  bacilli  suspended  in  the  air. 

The  exact  result,  of  course,  varies  in  different  animals  and 
according  to  the  method  of  inoculation,  but  we  may  state 
generally  that  when  introduced  into  the  tissues  of  a  susceptible 
animal,  the  bacilli  produce  locally  the  lesions  above  described, 
terminating  in  caseation ;  that  there  occurs  a  tubercular  affection 
of  the  neighbouring  lymphatic  glands,  and  that  lastly  there 
may  be  a  rapid  extension  of  the  bacilli  to  other  organs  by  the 
blood  stream  and  the  production  of  general  tuberculosis.  Of 
the  animals  generally  used  for  the  purpose,  the  guinea-pig  is 
most  susceptible. 

When  a  guinea-pig  is  inoculated  subcutaneously  with  tubercle 
bacilli  from  a  culture,  or  with  material  containing  them,  such  as 
phthisical  sputum,  a  local  swelling  gradually  forms  which  is 
usually  well  marked  about  the  tenth  day.  This  swelling  becomes 
softened  and  caseous,  and  may  break  down,  leading  to  the 
formation  of  an  irregularly  ulcerated  area  with  caseous  lining. 
The  lymphatic  glands  in  relation  to  the  parts  can  generally  be 
found  to  be  enlarged  and  of  somewhat  firm  consistence,  about 
the  end  of  the  second  or  third  week.  Later,  in  them  also  caseous 
change  occurs,  and  a  similar  condition  may  spread  to  other 
groups  of  glands  in  turn,  passing  also  to  those  on  the  other  side 
of  the  body.  During  the  occurrence  of  these  changes,  the  animal 
loses  weight,  gradually  becomes  .cachectic,  and  ultimately  dies, 
sometimes  within  six  weeks,  sometimes  not  for  two  or  three 
months.  Post  mortem,  in  addition  to  the  local  and  glandular 
changes,  an  acute  tuberculosis  is  usually  present,  the  spleen 
being  specially  affected.  This  organ  is  swollen,  and  is  studded 
throughout  by  numerous  tubercle  nodules,  which  may  be  minute 
and  grey,  or  larger  and  of  a  yellowish  tint.  If  death  has  been 
lono;  delayed,  calcification  may  have  occurred  in  some  of  the 
ii  Mlules.  Tubercle  nodules,  though  rather  less  numerous,  are 
also  present  in  the  liver  and  in  the  lungs,  the  nodules  in  the 
latti-r  organs  being  usually  of  smaller  size  though  occasionally  in 
large  numbers.  The  extent  of  the  general  infection  varies; 
-"inetimes  the  chronic  glandular  changes  constitute  the  out- 
standing feature.  Statements  as  to  differences  in  the  pathogenic 
effects  of  bacilli  from  human  and  bovine  sources  will  be  found 
below  (p.  274). 
18 


274  TUBERCULOSIS 

Varieties  of  Tuberculosis.  1.  Human  and  Bovine  Tuberculosis. 
—Up  till  recent  years  it  was  generally  accepted  that  all 
mammalian  tuberculosis  was  due  to  the  same  organism,  and, 
in  particular,  that  tuberculosis  could  be  transmitted  from  the 
ox  to  the  human  subject.  The  matter  became  one  of  special 
interest  owing  to  Koch's  address  at  the  Tuberculosis  Congress 
in  1901,  in  which  he  stated  his  conclusion  that  human  and 
bovine  tuberculosis  are  practically  distinct,  and  that  if  a 
susceptibility  of  the  human  subject  to  the  latter  really  exists, 
infection  is  of  very  rare  occurrence, — so  rare  that  it  is  not 
necessary  to  take  any  measures  against  it.  Previously  to  this, 
Theobald  Smith  had  pointed  out  differences  between  mammalian 
and  bovine  tubercle  bacilli,  the  most  striking  being  that  the 
latter  possess  a  much  higher  virulence  to  the  guinea-pig,  rabbit, 
and  other  animals,  and  in  particular  that  human  tubercle  bacilli, 
on  inoculation  into  oxen,  produce  either  no  disease  or  only  local 
lesions  without  any  dissemination.  Koch's  conclusions  were 
based  chiefly  on  the  result  of  his  inoculations  of  the  bovine 
species  with  human  tubercle  bacilli,  the  result  being  confirmatory 
of  Smith's,  and  also  on  the  supposition  that  infection  of  the 
human  subject  through  the  intestine  is  of  very  rare  occurrence. 

Since  the  time  of  Koch's  communication  an  enormous  amount 
of  work  has  been  done  on  this  subject,  and  Commissions  of 
inquiry  have  been  appointed  in  various  countries.  We  may 
summarise  the  chief  facts  which  have  been  established. 
Practically  all  observers  are  agreed  that  there  are  two  chief 
types  of  tubercle  bacilli,  which  differ  both  in  their  cultural 
characters  and  in  their  virulence — a  bovine  type  and  a  human 
type.  The  bacilli  of  the  bovine  type,  when  cultivated,  are  shorter 
and  thicker  and  more  regular  in  size ;  whilst  their  growth  on 
various  culture  media  is  scantier  than  that  of  the  human  type. 
From  the  latter  character  the  British  Royal  Commission  have 
applied  the  term  dysgonic  to  the  bovine  and  eugonic  to  the 
human  type.  As  already  stated,  there  is  also  a  great  difference 
in  virulence  towards  the  lower  animals,  the  bacillus  from  the  ox 
having  a  much  higher  virulence.  This  organism  when  injected 
in  suitable  quantities  into  the  ox  produces  a  local  tubercular 
lesion,  which  is  usually  followed  by  a  generalised  and  fatal 
tuberculosis ;  whereas  injection  of  human  tubercle  bacilli  pro- 
duces no  more  than  a  local  lesion,  which  undergoes  retrogression. 
(In  certain  experiments,  e.g.  those  of  Delepine,  Hamilton,  and 
Young,  general  tuberculosis  has  "been  produced  in  the  bovine 
species  by  tubercle  bacilli  from  the  human  subject,  but  these 
results  are  exceptional.)  Corresponding  differences  come  out 


VARIETIES  OF  TUBERCULOSIS  275 

in  the  case  of  the  rabbit;  in  fact,  intravenous  injection  of 
suitable  quantities  in  this  animal  is  the  readiest  method  of 
distinguishing  the  two  types — an  acute  tuberculosis  resulting 
with  the  bovine,  but  not  with  the  human  type.  In  guinea-pigs 
and  monkeys  a  generalised  tuberculosis  may  result  from  sub- 
cutaneous injection  of  bacilli  of  the  human  type,  but  in  this 
case  also  the  -difference  in  favour  of  the  greater  virulence  of  the 
bovine  type  is  made  out.  With  regard  to  the  distribution  of 
the  two  tyiws  of  organisms,  it  may  be  stated  that,  so  far  as  we 
know,  the  bacillus  obtained  from  bovine  tuberculosis  is  always 
of  the  bovine  type,  and  the  same  may  be  said  to  be  true  of 
tuberculosis  in  pigs ;  in  fact  this  seems  to  be  the  prevalent 
<>ri:aiii>!it  in  animal  tuberculosis.  In  human  tuberculosis  the 
bacilli  in  a  large  majority  of  the  cases  are  of  the  human  type ; 
but,  on  the  other  hand,  in  a  certain  proportion  bacilli  of  the 
bovine  type  are  present,  the  bacilli  when  cultivated  being 
indistinguishable  by  any  means  at  our  disposal  from  those 
obtained  from  bovine  tuberculosis.  The  Royal  Commission 
found  the  bovine  type  in  14  out  of  60  cases  of  human 
tuberculosis — a  somewhat  higher  proportion  than  has  been 
obtained  by  most  other  investigators — and  in  all  of  these, 
with  one  exception,  the  bacilli  were  obtained  either  from  caseous 
cervical  glands,  or  from  the  lesions  of  primary  abdominal  tuber- 
culosis, that  is  from  cases  where  there  was  evidence  of  infection 
by  alimentation.  It  is  also  to  be  noted  that  almost  all  the 
tubercular  lesions  from  which  the  bovine  type  has  been  obtained 
have  btrn  in  children.  The  general  result  accordingly  is  that 
bovine  tubercle  bacilli  are  present  in  a  certain  proportion  of 
caaea  of  tuberculosis  in  young  subjects,  and  that  these  are 
r-pccially  cases  where  infection  by  the  alimentary  canal  has 
occurred.  It  must  thus  be  held  as  established  that  tuberculosis 
is  transmissible  from  the  ox  to  man,  and  that  the  milk  of 
tubercular  cows  is  a  common  vehicle  of  transmission. 

Although  most  of  the  bacilli  which  have  been  cultivated 
correspond  to  one  of  the  two  types,  as  above  described,  it  is 
also  to%be  noted  that  intermediate  varieties  are  met  with.  It 
has  also  been  found  that  the  type  characters  of  the  bacillus  are 
not  constant.  Various  observers  have  found  it  possible  to 
modify  bacilli  of  the  human  type  by  passing  them  through  the 
bodies  of  certain  animals,  e.y.  guinea-pigs,  sheep,  and  goats,  So 
that  they  acquire  the  characters  of  bovine  bacilli.  In  view 
of  these  facts,  it  is  probable  that  bovine  bacilli  will  undergo 
corresponding  modifications  in  the  tissues  of  the  human  subject 
— what  period  of  time  is  necessary  for  such  a  change  we 


276  TUBERCULOSIS 

cannot  say.  It  is  thus  possible  that  the  cases  of  human  tuber- 
culosis from  which  the  bovine  type  has  been  obtained  do  not 
represent  the  full  number  where  infection  from  the  ox  has 
occurred.  It  is  quite  likely  that  although  the  bovine  bacilli 
are  more  virulent  to  the  lower  animals  than  the  human  bacilli 
are,  this  does  not  also  hold  in  the  case  of  the  human  subject. 
In  fact,  the  comparative  chronicity  of  the  primary  abdominal 
lesions  in  children,  in  the  first  instance,  would  point  rather  to  a 
low  order  of  virulence  towards  the  human  subject.  We  may 
also  add  that  there  are  cases,  notably  those  of  Ravenel,  in 
which  accidental  inoculation  of  the  human  subject  with  bovine 
tubercle  has  resulted  in  the  production  of  tuberculosis. 

2.  Avian  Tuberculosis. — In  the  tubercular  lesions  in  birds 
there  are  found  bacilli  which  correspond  in  their  staining  re- 
actions and  in  their  morphological  characters  with  those  in 
mammals,  but  differences  are  observed  in  cultures,  and  also  on 
experimental  inoculation.  These  differences  were  first  described 
by  Maffucci  and  by  Rivolta,  but  special  attention  was  drawn  to 
the  subject  by  a  paper  read  by  Koch  at  the  International  Medical 
Congress  in  1890.  Koch  stated  that  he  had  failed  to  change 
the  one  variety  of  tubercle  bacillus  into  the  other,  though  he  did 
not  conclude  therefrom  that  they  were  quite  distinct  species. 
The  following  points  of  difference  may  be  noted : — 

On  glycerin  agar  and  on  serum,  the  growth  of  tubercle  bacilli  from 
birds  is  more  luxuriant,  has  a  moister  appearance  (Fig.  79,  C),  and, 
moreover,  takes  place  at  a  higher  temperature,  43*5°  C.,  than  is  the  case 
with  mammalian  tubercle  bacilli.  Experimental  inoculation  brings  out 
even  more  distinct  differences.  Tubercle  bacilli  derived  from  the  human 
subject,  for  example,  when  injected  into  birds,  usually  fail  to  produce 
tuberculosis,  whilst  those  of  avian  origin  very  readily  do  so.  Birds  are 
also  very  susceptible  to  the  disease  when  fed  with  portions  of  the  organs 
of  birds  containing  tubercle  bacilli,  but  they  can  consume  enormous 
quantities  of  phthisical  sputum  without  becoming  tubercular  (Strauss, 
Wurtz,  Nocard).  No  doubt,  on  the  other  hand,  there  are  cases  on  record 
in  which  the  source  of  infection  of  a  poultry-yard  has  apparently  been 
the  sputum  of  phthisical  patients.  Again,  tubercle  bacilli  cultivated  from 
birds  have  not  the  same  effect  on  inoculation  of  mammals  as  ordinary 
tubercle  bacilli  have.  When  guinea-pigs  are  inoculated  subcutaneously 
they  usually  resist  infection,  though  occasionally  a  fatal  result'  follows. 
In  the  latter  case,  usually  no  tubercles  visible  to  the  naked  eye  are  found, 
but  numerous  bacilli  may  be  present  in  internal  organs,  especially  in  the 
spleen,  which  is  .much  swollen.  Further,  intravenous  injection  even  of 
large  quantities  of  avian  tubercle  bacilli,  in  the  case  of  dogs,  leads  to  no 
effect,  whereas  ordinary  tubercle  bacilli  produce  acute  tuberculosis.  [The 
rabbit,  on  the  other  hand,  is  comparatively  susceptible  to  avian  tuber- 
culosis (Nocard).] 

There  is,  therefore,  abundant  evidence  that  the  bacilli  derived 


VARIETIES  OF  TUBERCULOSIS  277 

from  the  two  classes  of  animals  show  important  differences,  and, 
reasoning  from  analogy,  we  might  infer  that  probably  the  human 
.subject  also  would  be  little  susceptible  to  infection  from  avian 
tuberculosis.  The  question  remains,  are  these  differences  of  a 
permanent  character  1  The  matter  seems  conclusively  settled  by 
the  experiments  of  Nocard,  in  which  mammalian  tubercle  bacilli 
have  been  made  to  acquire  all  the  characters  of  those  of  avian 
origin.  The  method  adopted  was  to  place  bacilli  from  human 
tuberculosis  in  small  collodion  sacs  (v.  p.  144)  containing  bouillon, 
and  then  to  insert  each  sac  in  the  peritoneal  cavity  of  a  fowl. 
The  sacs  were  left  in  situ  for  periods  of  from  four  to  eight 
months.  They  were  then  removed,  cultures  were  made  from 
their  contents,  fresh  sacs  were  inoculated  from  these  cultures 
and  introduced  into  other  fowls.  In  such  conditions  the  bacilli 
are  subjected  only  to  the  tissue  juices,  the  wall  of  the  sac  being 
impervious  both  to  bacilli  and  to  leucocytes,  etc.  After  one 
sojourn  of  this  kind,  and  still  more  so  after  two,  the  bacilli  are 
found  to  have  acquired  some  of  the  characters  of  avian  tubercle 
bacilli,  but  are  still  non-virulent  to  fowls.  After  the  third 
sojourn,  however,  they  have  acquired  this  property,  and  produce 
in  fowls  the  same  lesions  as  bacilli  derived  from  avian  tuber- 
culosis. It  therefore  appears  that  the  bacilli  of  avian  tuber- 
culosis are  not  a  distinct  and  permanent  species,  but  a  variety 
which  has  been  modified  by  growth  in  the  tissues  of  the  bird. 
It  is  also  interesting  to  note  that  Rabinowitch  has  cultivated 
tubercle  bacilli  of  the  mammalian  type  from  some  cases  of  tuber- 
culosis in  parrots  kept  in  confinement. 

3.  Tuberculosis  in  the  Fish. — Bataillon,  Dubard,  and  Terre 
cultivated  from  a  tubercle-like  disease  in  a  carp,  a  bacillus 
which,  in  staining  reaction  and  microscopic  characters,  closely 
agrees  with  the  tubercle  bacillus.  The  lesion  with  which  it 
was  associated  was  an  abundant  growth  of  granulation  tissue  in 
which  numerous  giant-cells  were  present.  It  forms,  however, 
luxuriant  growth  at  the  room  temperature,  the  growth,  being 
thick  and  moist  like  that  of  avian  tubercle  bacilli  (Fig.  84,  c). 
Growth  does  not  occur  at  the  body  temperature,  though  by 
gradual  acclimatisation  a  small  amount  of  growth  has  been 
obtained  up  to  36°  C.  Furthermore,  the  organism  appears  to 
undergo  no  multiplication  when  injected  into  the  tissues  of 
mammals,  and  attempts  to  modify  this  characteristic  have  so 
far  been  unsuccessful.  Weber  and  Taute  have  cultivated  this 
organism  from  mud,  and  also  from  organs  of  healthy  frogs.  It 
is  thus  probably  to  be  regarded  as  a  saprophyte  which  is  only 
occasionally  associated  with  disease  in  the  fish.  According  to 


278  TUBERCULOSIS 

the  results  of  different  experimenters,  it  is  possible  to  modify 
human  tubercle  bacilli  by  allowing  them  to  sojourn  in  the 
tissues  of  cold-blooded  animals,  e.g.  the  frog,  blind-worm,  etc., 
so  that  they  flourish  at  lower  temperatures.  These  results  have, 
however,  been  recently  called  in  question,  as  it  has  been  stated 
the  organisms  obtained  were  not  modified  tubercle  bacilli,  but 
other  acid-fast  bacilli  which  may  be  found  in  the  tissues  of 
normal  cold-blooded  animals.  This  question  must  accordingly 
be  considered  still  an  open  one. 

All  the  above  facts  taken  together  indicate  that  tubercle 
bacilli  may  become  modified  in  relative  virulence  and  in  con- 
ditions of  growth  by  sojourn  in  the  tissues  of  various  animals. 
This  modification  appears  slight,  though  of  definite  character  in 
the  case  of  bovine  tuberculosis,  more  distinct  in  the  case  of 
avian  tuberculosis,  and  much  more  marked,  if  not  permanent,  in 
the  case  of  fish  tuberculosis,  that  is,  of  course,  in  their  relations 
to  the  bacilli  from  the  human  subject. 

Other  Acid-fast  Bacilli. — Within  recent  years  a  number  of 
bacilli  presenting  the  same  staining  reaction  as  the  tubercle 
bacilli  have  been  discovered.  Such  bacilli  have  a  comparatively 
wide  distribution  in  nature,  as  they  have  been  obtained  from 
various  species  of  grass,  from  butter  and  milk,  from  manure, 
and  from  the  surfaces  of  animal  bodies.  Microscopically,  they 
agree  more  or  less  closely  with  tubercle  bacilli,  though  most  of 
them  are  shorter  and  plumper ;  many  of  them  show  filamentous 
and  branching  forms  under  certain  conditions  of  culture.  More- 
over, on  injection,  they  produce  granulation-tissue  nodules  which 
may  closely  resemble  tubercles,  although  on  the  whole  there  is 
a  greater  tendency  to  softening  and  suppuration,  and  usually  the 
lesions  are  localised  to  the  site  of  inoculation.  The  most  im- 
portant point  of  distinction  is  the  fact  that  their  multiplication 
on  artificial  media  is  much  more  rapid,  growth  usually  being 
visible  within  forty-eight  hours  .and  often  within  twenty-four 
hours  at  37°  C.  Furthermore,  in  most  instances  growth  occurs 
at  the  room  temperature.  The  general  character  of  the  cultures 
in  this  group  is  a  somewhat  irregular  layer,  often  with  wrinkled 
surface,  dry  or  moist  in  appearance,  and  varying  in  tint  from 
white  to  yellow  or  reddish  brown.  The  number  of  such  organ- 
isms is  constantly  being  added  to,  but  the  following  may  be 
mentioned  as  examples  : — 

Moellers  Grass  Bacilli  I.  and  II. — The  former  was  found  in  infusions 
of  Timothy-grass  (Phleum  pratense).  It  is  extremely  acid-fast,  morpho- 
logically resembles  the  tubercle  bacillus,  and  in  cultures  may  show  club 
formation  and  branching.  The  lesions  produced  closely  resemble 


OTHER  ACID-FAST  BACILLI  279 

tubercles.  The  colonies,  visible  in  thirty-six  hours,  are  scale-like  and  of 
^ivyish-whitc  colour  (Fig.  84,  a}.  Moeller's  bacillus  II.  was  obtained 
from  the  dust  of  a  hay-loft.  The  colonies  at  first  are  moist  and  some- 
what tenacious,  but  afterwards  run  together,  and  are  of  a  dull  yellowish 
colour.  The  general  results  of  inoculation  resemble  those  of  grass 
bacillus  I.,  but  are  less  marked.  Moeller  also  obtained  a  similar  organism 
from  milk.  He  also  discovered  a  third  acid-fast  bacillus,  which  ho 
obtained  from  manure  and  therefore  called  the  "  Mistbacillus  "  (dung 
bacillus).  This  organism  has  analogous  characters,  though  presenting 
minor  differences.  It  also  has  pathogenic  effects. 

Petri  and  Rabinowitch  independently  cultivated  an  acid-fast  bacillus 
from   butter  ("butter  bacillus"),  in  which  it  occurs  with  comparative 


/*   * 


FIG.  83.— Moeller's  Timothy-grass  bacillus.  c 

From  a  culture  on  agar.  FIG.  84.— Cultures  of  acid-fast  bacilli 

Stained  with  carbol-fuchsin,   and    treated  grown  at  room  temperature, 

with  20  per  cent,  sulphuric  acid.  (rt)  Moeller's  Timothy-grass  bacillus  I. 

x  1000.  (&)  The  Petri-Rabinowitch  butter  bacillus, 

(c)  Bacillus  of  fish  tuberculosis. 

frequency.  The  organism  resembles  the  tubercle  bacillus,  although  it  is 
on  the  whole  shorter  and  thicker.  Its  lesions  closely  resemble  tuber- 
culosis, especially  when  injection  of  the  organism  is  made  into  the 
peritoneal  cavity  of  guinea-pigs,  along  with  butter,— the  method  usually 
adopted  in  searching  for  tubercle  bacilli  in  butter.  This  organism 
produces  pretty  rapidly  a  wrinkled  growth  (Fig.  84,  b)  not  unlike  that 
of  Moeller's  grass  bacillus  II.  Korn  has  also  obtained,  other  two  bacilli 
from  butter  which  he  holds  to  be  distinct  from  one  another  and  from 
Rabinowitch's  bacillus.  The  points  of  distinction  are  of  a  minor 
character.  Other  more  or  less  similar  bacilli  have  been  cultivated  by 
Tobler,  Coggi,  and  others.1 

Another  bacillus  of  considerable  interest  is  Johne's  bacillus  or  the 

i  For  further  details  on  this  subject,  vide  Potet,  Etudes  sur  les  bacilles  dites 
acidophiles.     Paris,  1902. 


280 


TUBERCULOSIS 


V- 


>r 


bacillus  of  "chronic  bovine  pseudo-tuberculous  enteritis,"  the  lesions 
produced  by  it  being  corrugated  thickenings  of  the  mucous  membrane, 
especially  of  the  small  intestine.  The  disease  has  now  been  observed  in 
various  countries,  and  several  cases  in  Britain  have  been  recorded  by 
M'Fadyean.  The  bacilli  occur  in  large  numbers  in  the  lesions,  and  can 
readily  be  found  in  scrapings  from  the  surface.  They  resemble  the 
tubercle  bacillus  in  appearance,  but  on  the  whole  are  rather  shorter  ;  they 
are  equally  acid-fast.  The  organism  has  not  yet  been  cultivated  outside 
the  body. 

Smegma  Bacillus. — This  organism  is  of  importance,  as  in  form  and 
staining  reaction  it  somewhat  resembles  the  tubercle  bacillus  and  may  be 

mistaken  for  it.      It  occurs 

^,—     /     •  often   in   large  numbers    in 

the  smegma  preeputiale  and 
in  the  region  of  the  external 
genitals,  especially  where 
there  is  an  accumulation  of 
fatty  matter  from  the  secre- 
tions. Morphologically  it  is 
a  slender,  slightly  curved 
organism,  like  the  tubercle 
bacillus,  but  usually  dis- 
tinctly shorter  (Fig.  85). 
Like  the  tubercle  bacillus,  it 
stains  with  some  difficulty 
and  resists  decolorisation 
with  strong  mineral  acids. 
Most  observers  ascribe  the 
latter  fact  to  the  fatty 
matter  with  which  it  is 
surrounded,  and  find  that 
if  the  specimen  is  treated 
with  alcohol  the  organ- 
ism is  easily  decolorised. 
Czaplewski,  however,  who 

claims  to  have  cultivated  it  on  various  media,  finds  that  in  culture  it 
shows  resistance  to  decolorisation  both  with  alcohol  and  with  acids,  and 
considers,  therefore,  that  the  reaction  is  not  due  to  the  surrounding  fatty 
medium.  We  have  found  that  in  smegma  it  can  be  readily  decolorised 
by  a  minute's  exposure  to  alcohol  after  the  usual  treatment  with  sulphuric 
acid,  and  thus  can  be  readily  distinguished  from  the  tubercle  bacillus. 
We,  moreover,  believe  that  minor  points  of  difference  in  the  microscopic 
appearances  of  the  two  organisms  are  quite  sufficient  to  make  the 
experienced  observer  suspicious  if  he  should  meet  with  the  smegma 
bacillus  in  urine,  and  lead  him  to  apply  the  decolorising  test.  Difficulty 
will  only  occur  when  a  few  scattered  bacilli  retaining  the  fuchsin  are 
found. 

Its  cultivation,  which  is  attended  with  some  difficulty,  was  first  effected 
by  Czaplewski.  On  serum  it  grows  in  the  form  of  yellowish -grey 
irregularly  rounded  colonies  about  1  mm.  in  diameter,  sometimes 
becoming  confluent  to  form  a  comparatively  thick  layer.  He  found  that 
it  also  grew  on  glycerin  agar  and  in  bouillon.  It  is  non -pathogenic  to 
various  animals  which  have  been  tested. 

Cowie  has  recently  found  that  acid- fast  bacilli  are  of  common  occur- 


FIG.  85. — Smegma  bacilli.     Film  preparation 

of  smegma. 
Ziehl-Neelsen  stain,      x  1000. 


ACTION  OF  DEAD  TUBERCLE  BACILLI        281 

rence  in  the  secretions  of  the  external  genitals,  mammae,  etc.,  in  certain 
of  the  lower  animals,  and  that  these  organisms  vary  in  appearance.  He 
considers  that  the  term  "smegma  bacillus"  probably  represents  a 
number  of  allied  species. 

The  question  may  be  asked — do  these  results  modify  the 
validity  of  the  staining  reaction  of  tubercle  bacilli  as  a  means  of 
diagnosis?  The  source  of  any  acid-fast  bacilli  in  question  is 
manifestly  of  importance,  and  it  may  be  stated  that  when  these 
have  been  obtained  from  some  source  outside  the  body,  or  where 
contamination  from  without  has  been  possible,  their  recognition 
as  tubercle  bacilli  cannot  be  established  by  microscopic  examina- 
tion alone.  In  the  case  of  material  coming  from  the  interior  of 
the  body,  however, — sputum,  etc., — the  condition  must  be  looked 
on  as  different,  and  although  an  acid-fast  bacillus  (not  tubercle) 
has  been  found  by  Rabinowitch  in  a  case  of  pulmonary  gangrene, 
we  have  no  sufficient  data  for  saying  that  acid-fast  bacilli  other 
than  the  tubercle  bacillus  flourish  within  the  tissues  of  the  hwnian 
body,  except  in  such  rare  instances  as  to  be  practically  negligible. 
(To  this  statement  the  case  of  the  leprosy  bacillus  is  of  course 
an  exception.)  Accordingly,  up  till  now,  the  microscopic  ex- 
amination of  sputum,  etc.,  cannot  be  said  to  have  its  validity 
shaken,  and  we  have  the  results  of  enormous  clinical  experience 
that  such  examination  is  of  practically  unvarying  value.  Never- 
theless the  facts  established  with  regard  to  other  acid-fast  bacilli 
must  be  kept  carefully  in  view,  and  great  care  must  be  exercised 
when  only  one  or  two  bacilli  are  found,  especially  if  they  deviate 
in  their  morphological  characters  from  the  tubercle  bacillus. 

Action  of  dead  Tubercle  Bacilli. — The  remarkable  fact  has 
been  established  by  independent  investigators,  that  tubercle 
bacilli  in  the  dead  condition,  when  introduced  into  the  tissues 
in  sufficient  numbers,  can  produce  tubercle-like  nodules.  Prudden 
and  Hodenpyl,  by  intravenous  injection  in  rabbits  of  cultures 
sterilised  by  heat,  produced  in  the  lungs  small  nodules  in  which 
giant-cells,  but  no  caseation,  were  occasionally  present,  and 
which  were  characterised  by  more  growth  of  fibrous  tissue  than 
in  ordinary  tubercle.  The  subject  was  very  fully  investigated 
with  confirmatory  results  by  Straus  and  Gamaleia,  who  found 
that,  if  the  number  of  bacilli  introduced  into  the  circulation  were 
large,  there  resulted  very  numerous  tubercle  nodules  with  well- 
formed  giant-cells,  and  occasionally  traces  of  caseation.  The 
bacilli  can  be  well  recognised  in  the  nodules  by  the  ordinary 
staining  method.  In  these  experiments  the  bacilli  were  killed 
by  exposure  to  a  temperature  of  115°  C.  for  ten  minutes  before 
being  injected.  Similar  nodules  can  be  produced  by  intra- 


282  TUBERCULOSIS 

peritoneal  injection.  Subcutaneous  injection,  on  the  other 
hand,  produces  a  local  abscess,  but  in  this  case  no  secondary 
tubercles  are  found  in  the  internal  organs.  Further,  in  many 
of  the  animals  inoculated  by  the  various  methods,  a  condition  of 
marasmus  sets  in  and  gradually  leads  to  a  fatal  result,  there 
being  great  emaciation  before  death.  These  experiments,  which 
have  been  confirmed  by  other  observers,  show  that  even  after 
the  bacilli  are  dead  they  preserve  their  staining  reactions  in 
the  tissues  for  a  long  time,  and  also  that  there  are  apparently 
contained  in  the  bodies  of  the  dead  bacilli  certain  substances 
which  act  locally,  producing  proliferative  and,  to  a  less  extent, 
degenerate  changes,  and  which  also  markedly  affect  the  general 
nutrition.  S.  Stockman  has  found  that  an  animal  inoculated 
with  large  numbers  of  dead  tubercle  bacilli  afterwards  gives  the 
tuberculin  reaction. 

Practical  Conclusions. — From  the  facts  above  stated  with 
regard  to  the  conditions  of  growth  of  the  tubercle  bacilli,  their 
powers  of  resistance,  and  the  paths  by  which  they  can  enter  the 
body  and  produce  disease  (as  shown  by  experiment),  the  manner 
by  which  tuberculosis  is  naturally  transmitted  can  be  readily 
understood.  Though  the  experiments  of  Sander  show  that 
tubercle  bacilli  can  multiply  on  vegetable  media  to  a  certain 
extent  at  warm  summer  temperature,  it  is  doubtful  whether  all 
the  conditions  necessary  for  growth  are  provided  to  any  extent 
in  nature.  At  any  rate,  the  great  multiplying  ground  of  tubercle 
bacilli  is  the  animal  body,  and  tubercular  tissues  and  secretions 
containing  the  bacilli  are  the  chief,  if  not  the  only,  means  by 
which  the  disease  is  spread.  The  tubercle  bacilli  leave  the  body 
in  large  numbers  in  the  sputum  of  phthisical  patients,  and  when 
the  sputum  becomes  dried  and  pulverised  they  are  set  free  in 
the  air.  Their  powers  of  resistance  in  this  condition  have  already 
been  stated.  As  examples  of  the  extent  to  which  this  takes 
place,  it  may  be  said  that  their  presence  in  the  air  of  rooms 
containing  phthisical  patients  has  been  repeatedly  demonstrated. 
Williams  placed  glass  plates  covered  with  glycerine  in  the 
ventilating  shaft  of  the  Brompton  Hospital,  and  after  five  days 
found,  by  microscopic  examination,  tubercle  bacilli  on  the  surface, 
whilst  Klein  found  that  guinea-pigs  kept  in  the  ventilating  shaft 
became  tubercular.  Cornet  produced  tuberculosis  in  rabbits  by 
inoculating  them  with  dust  collected  from  the  walls  of  a  con- 
sumptive ward.  Tubercle  bacilli  are  also  discharged  in  consider- 
able quantities  in  the  urine  in  tubercular  disease  of  the  urinary 
tract,  and  also  by  the  bowel  when  there  is  tubercular  ulceration ; 
but,  so  far  as  the  human  subject  is  concerned,  the  great  means 


PRACTICAL  CONCLUSIONS  283 

of  disseminating  the  bacilli  in  the  outer  world  is  dried  phthisical 
sputum,  and  the  source  of  danger  from  this  means  can  scarcely 
be  overestimated.  Every  phthisical  patient  ought  to  be  looked 
upon  as  a  fruitful  source  of  infection  to  those  around,  and  should 
only  expectorate  on  pieces  of  rag  which  are  afterwards  to  be 
burnt,  or  into  special  receptacles  which  are  to  be  then  sterilised 
either  by  boiling  or  by  the  addition  of  a  5  per  cent,  solution  of 
carbolic  acid. 

Another  great  source  of  infection  is  in  all  probability  the 
milk  of  cows  affected  with  tuberculosis  of  the  udder.  In  such 
cases  the  presence  of  tubercle  bacilli  in  the  milk  can  usually  be 
readily  detected  by  centrifugalising  it,  and  then  examining  the 
dei>osit  microscopically,  or  by  inoculating  an  animal  with  it.  As 
pointed  out  by  Woodhead  and  others,  the  milk  from  cows  thus 
affected  is  probably  the  great  source  of  tabes  mesenterica,  which 
is  so  common  in  young  subjects.  In  these  cases  there  may  be 
tubercular  ulceration  of  the  intestine,  or  it  may  be  absent. 
Woodhead  found  that  out  of  127  cases  of  tuberculosis  in  children, 
the  mesenteric  glands  showed  tubercular  affection  in  100,  and 
that  there  was  ulceration  of  the  intestine  in  43.  It  is  especially 
in  children  that  this  mode  of  infection  occurs,  as  in  the  adult 
ulceration  of  the  intestine  is  rare  as  a  primary  infection,  though 
it  is  common  in  phthisical  patients  as  the  result  of  infection  by 
the  bacilli  in  the  sputum  which  has  been  swallowed.  There  is 
less  risk  of  infection  by  means  of  the  flesh  of  tubercular  animals, 
for,  in  the  first  place,  tuberculosis  of  the  muscles  of  oxen  being 
very  rare,  there  is  little  chance  of  the  bacilli  being  present  in  the 
flesh  unless  the  surface  has  been  smeared  with  the  juice  of  the 
tubercular  organs,  as  in  the  process  of  cutting  up  the  parts  ;  and, 
in  the  second  place,  even  when  present  they  will  be  destroyed  if 
the  meat  is  thoroughly  cooked. 

We  may  state,  therefore,  that  the  two  great  modes  of  infection 
are  by  inhalation,  and  by  ingestion,  of  tubercle  bacilli.  By  the 
former  method  the  tubercle  bacilli  will  in  most  cases  be  derived 
from  the  human  subject ;  in  the  latter,  probably  from  tubercular 
cows,  though  inhaled  tubercle  bacilli  may  also  be  swallowed  and 
contamination  of  food  by  tubercular  material  from  the  human 
subject  may  occur.  Alike  when  inhaled  and  when  ingested, 
tubercle  bacilli  may  lodge  about  the  pharynx  and  thus  come  to 
infect  the  pharyngeal  lymphoid  tissue,  tonsils,  etc.,  tubercular 
lesions  of  these  parts  being  much  more  frequent  than  was 
formerly  supposed.  Thence  the  cervical  lymphatic  glands  may 
become  infected,  and  afterwards  other  groups  of  glands,  bones, 
or  joints,  and  internal  organs. 


284  TUBERCULOSIS 

The  Specific  Reactions  of  Tubercle  Bacillus. — The  tubercle 
bacillus  belongs  to  the  group  of  organisms  which  do  not  secrete 
soluble  toxins  into  the  media  in  which  they  are  growing.  It 
shares  with  other  members  of  the  group  the  capacity  to  induce 
serious  changes  in  the  metabolism  of  an  animal.  As,  in  other 
similar  cases,  we  are  in  the  dark  as  to  how  these  changes  come 
about,  and  thus  can  only  summarise  the  chief  effects  which,  by 
present  methods,  can  be  detected  as  occurring  in  the  bodies  of 
infected  animals.  These  effects  which,  it  may  be  remarked,  are 
of  value  in  the  diagnosis  of  tubercular  affections,  consist  on  the 
one  hand  (a)  of  certain  phenomena  of  supersensitiveness,  and  on 
the  other  (6)  of  certain  changes  in  the  blood  serum  of  tubercular 
patients  resulting  from  reactions  of  immunity.  The  former  are 
seen  when  the  bacilli  or  substances  artificially  derived  from  their 
bodies  are  introduced  into  the  tissues  of  those  suffering  from 
tuberculosis,  and  were  first  demonstrated  by  Koch  in  his  work 
on  tuberculin.  In  recent  times,  examples  of  similar  effects  are 
the  ophthalmic  reaction  of  Calmette  and  the  cutaneous  reaction 
of  von  Pirquet.  The  changes  in  the  blood  serum  of  infected 
persons  depend  on  the  presence  of  anti-substances  in  the  blood. 
These  may  be  of  the  nature  of  (a)  immune  bodies  which  lead  to 
fixation  of  complement,  and  (b)  precipitins,  (c)  agglutinins,  (d) 
opsonins.  These  may  now  be  severally  discussed  in  detail. 

(1)  Phenomena  of  Supersensitiveness.  (a)  Koctis  Old  Tuber- 
culin.— Koch  (1890-1)  stated  that  if  in  a  guinea-pig  suffering 
from  the  effects  of  a  subcutaneous  inoculation  with  tubercle 
bacilli,  a  second  subcutaneous  inoculation  of  tubercle  bacilli  was 
practised  in  another  part  of  the  body,  superficial  ulceration 
occurred  in  the  primary  tubercular  nodule,  the  wound  healed, 
and  the  animal  did  not  succumb  to  tuberculosis.  This  reaction 
was  further  studied  by  means  of  tuberculin,  which  consisted  of 
a  concentrated  glycerin  bouillon  culture  of  tubercle  in  which  the 
bacilli  had  been  killed  by  heat.  Its  essential  components  pro- 
bably were  the  dead  and  often  macerated  bacilli  and  the 
substances  indestructible  by  boiling  which  existed  in  these 
bacilli,  or  which  were  formed  during  their  growth.  The  in- 
jection of  '25  c.c.  of  tuberculin  into  a  healthy  man  causes,  in 
from  three  to  four  hours,  malaise,  tendency  to  cough,  laboured 
breathing,  and  moderate  pyrexia ;  all  of  which  pass  off  in 
twenty-four  hours.  The  subcutaneous  injection,  however,  of 
"01  c.c.  into  a  tubercular  person  gives  rise  to  similar  symptoms 
(now  known  as  the  tuberculin  reaction),  but  in  a  much  more 
aggravated  form,  and  in  addition  there  occurs  around  any 
tubercular  focus  great  inflammatory  reaction,  resulting  in  necrosis 


PHENOMENA  OF  SUPERSENSITIVENESS       285 

and  a  casting  off  of  the  tubercular  mass,  when  this  is  possible, 
as  for  instance  in  the  case  of  lupus.  The  bacilli  are,  it  was 
shown,  not  killed  in  the  process. 

The  hopes  which  the  introduction  of  tuberculin  raised,  that  a 
curative  agent  against  tuberculosis  had  been  discovered,  were 
soon  found  not  to  be  justified.  It  was  very  difficult  to  see  how 
the  necrosed  material  which  was  produced  and  which  contained 
the  still  living  bacilli,  could  be  got  rid  of  either  naturally,  as 
would  be  necessary  in  the  case  of  a  small  tubercular  deposit  in 
a  lung  or  a  lymphatic  gland,  or  artificially,  as  in  a  complicated 
joint-cavity  where  surgical  interference  could  be  undertaken. 
Not  only  so,  but  the  ulceration  which  might  be  the  sequel  of  the 
necrosis  appeared  to  open  a  path  for  fresh  infection.  Soon  facts 
were  reported  which  justified  these  criticisms.  Cases  where 
rapid  acute  tubercular  conditions  ensued  on  the  use  of  tuberculin 
were  reported,  and  in  a  few  months  the  treatment  was  practically 
abandoned. 

(b)  The  Cutaneous  Tuberculin  reaction  of  von  Pirquet  and 
tJie  Op/tt/tal  mo-reaction  of  Calmette. — In  recent  times  the  diagnosis 
of  tuberculosis  has  been  considerably  aided  by  the  introduction 
of  these  two  tests.  Both  are  essentially  of  the  same  nature,  and 
depend  like  the  original  tuberculin  reaction  on  the  sensitiveness 
of  the  tissues  of  tubercular  patients  to  tuberculin. 

The  cutaneous  test  is  carried  out  as  follows  :  The  skin,  usually 
that  of  the  flexor  aspect  of  the  forearm,  is  well  cleansed  with 
ether  and  then  allowed  to  dry.  Two  drops  of  tuberculin  are 
placed  on  the  prepared  surface  about  four  inches  apart,  and  then 
midway  between  the  two  drops  a  small  spot  is  scarified  with  a 
small  metal  bore  constructed  for  the  purpose.  This  serves  as  a 
control,  any  reaction  which  follows  in  it  being  merely  a  traumatic 
one.  Similar  scarification  is  effected  through  the  drops  of 
tuberculin,  so  that  the  scarified  spots  are  exposed  to  its  action. 
Small  portions  of  cotton  wool  are  placed  over  the  drops  to 
prevent  the  tuberculin  from  running  off,  and  the  latter  is  allowed 
to  act  for  ten  minutes.  After  that  time  the  cotton  wool  is 
removed  ;  no  dressing  is  required.  In  the  process  of  scarification 
only  the  epidermis  should  be  injured  and  blood  should  not  be 
drawn.  The  "old"  tuberculin  of  Koch  is  that  used.  In  the 
case  of  a  positive  reaction  an  inflammatory  redness  and  swelling 
make  their  appearance  round  the  sites  of  tuberculin  inoculation, 
generally  within  a  few  hours,  and  at  the  end  of  twenty-four  hours 
there  is  a  distinct  inflammatory  papule  about  half  an  inch  in 
diameter,  with  a  somewhat  paler  centre  like  a  spot  of  urticaria; 
sometimes  in  the  centre  there  are  minute  vesicles.  The  maximum 


286  TUBERCULOSIS 

effect  usually  occurs  within  forty- eight  hours,  and  after  that  time 
the  reaction  gradually  recedes.  Such  is  the  typical  reaction,  but 
of  course  slighter,  and  also  more  intense  reactions  are  met  with. 
In  a  negative  reaction  all  three  points  of  scarification  show 
merely  a  slight  traumatic  redness  which  soon  passes  off. 

For  the  ophthalmo-reaction  Calmette  uses  a  purified  tuberculin. 
The  tuberculin  is  prepared  as  in  Koch's  original  method,  and  is 
precipitated  with  95  per  cent,  alcohol;  the  precipitate  is  then 
dissolved  in  water.  This  process  is  repeated  other  two  times, 
and  the  final  precipitate  is  made  up  as  a  1  per  cent,  solution  in 
distilled  water.  For  use,  in  the  case  of  an  adult,  a  drop  of  this 
solution  is  placed  in  the  conjunctival  sac  and  the  fluid  allowed 
to  spread  over  the  surface  ;  for  children  about  half  this  quantity 
is  sufficient.  In  the  case  of  a  positive  reaction  the  ocular  con- 
junctiva is  congested,  the  lids  become  somewhat  swollen  and 
their  inner  surface  presents  a  bright  red  colour,  there  is  increased 
secretion  of  tears  and  a  varying  amount  of  fibrinous  exudation. 
The  reaction  usually  reaches  its  maximum  in  from  six  to  ten 
hours  after  the  instillation,  and  commences  to  pass  off"  in  from 
twenty-four  to  thirty-six  hours, — in  children  a  little  sooner. 

The  general  results  obtained  by  these  two  reactions  appear  to 
correspond  closely.  A  distinct  positive  result  obtained  by  either 
is  practically  conclusive  as  to  the  presence  of  a  tubercular  lesion. 
In  cases  of  latent  tuberculosis  the  reaction  is  sometimes  obtained, 
sometimes  not.  Again,  in  very  advanced  cases  of  tuberculosis, 
especially  a  short  time  before  death,  a  negative  result  may  be 
got  y  in  some  of  these  cases  v.  Pirquet  has  met  with  a  colourless 
papule  or  a  livid  spot  without  exudation,  conditions  which  he 
describes  as  indicating  a  "cachectic  reaction."  The  ophthalmo- 
reaction  is  the  more  easily  applied,  at  least  in  adults,  but  its  use 
is  contra-indicated  when  there  is  any  abnormal  condition  of  the 
conjunctiva.  Even  apart  from  this,  however,  inflammatory 
symptoms  of  disagreeable  severity  sometimes  supervene.  It 
should  also  be  noted  that  a  second  test  ought  not  to  be  applied 
to  the  same  eye,  as  the  first  may  produce  a  condition  of  super- 
sensibility  (p.  284).  V.  Pirquet  claims  for  his  method  that  in  the 
case  of  children  it  can  be  satisfactorily  carried  out  with  greater 
ease  than  the  ophthalmic  test. 

It  will  be  recognised  that  the  processes  underlying  the  original 
tuberculin  reaction  on  the  one  hand,  and  the  cutaneous  and 
ophthalmic  reactions  on  the  other,  are  analogous.  In  the  former 
there  is  the  occurrence  of  local  inflammation  with  metabolic 
changes  and  fever ;  in  the  latter,  of  mild  inflammatory  effects, — 
in  both  cases  the  phenomena  being  found  only  in  tubercular 


PHENOMENA  OF  SUPERSENSITIVENESS       287 

subjects.  The  original  explanation  given  by  Koch  of  the 
tuberculin  reaction  was  that  the  essential  constituent  of  tuber- 
culin being  toxic  products  of  the  tubercle  bacillus,  the  action 
of  these  was  superadded  to  the  toxins  produced  at  the  focus  of 
infection.  The  combined  action  of  the  toxins  from  these  two 
sources  caused  a  rapid  necrosis  of  the  newly  formed  cells,  and 
opened  the  way  for  the  dead  tissue  being  rapidly  cast  off.  This 
explanation  was,  however,  not  generally  accepted,  for  it  was 
found  that  other  substances,  such  as  albumoses,  when  injected 
into  animals  suffering  from  local  tuberculosis,  gave  rise  to  modified 
effects  of  the  same  kind  as  those  produced  by  tuberculin.  This 
dissatisfaction  with  regard  to  the  original  explanation  is  ac- 
centuated by  a  consideration  of  the  effects  seen  in  the  Calmette 
and  v.  Pirquet  tests,  as  these  clearly  indicate  that  the  sensitive- 
ness in  a  tubercular  subject  is  not  confined  to  tissues  actually 
affected  with  the  tubercular  process,  but  is  also  manifested  in 
parts  of  the  body  distant  from  the  site  of  actual  infection. 

Further,  it  has  been  found,  first,  that  the  injection  of  tuber- 
culin directly  into  a  tubercular  focus  does  not  produce  the 
tuberculin  reaction  (a  fact  which  militates  against  the  idea  of 
concentrations  of  toxins),  and,  secondly,  that  the  injection  of 
living  or  dead  tubercle  bacilli  into  healthy  animals  produces 
anaphylactic  phenomena  similar  to  those  originated  by  foreign 
albumins  generally.  At  the  present  time,  therefore,  although 
no  full  explanation  can  be  given  of  the  tuberculin  reaction  and 
of  similar  reactions,  it  is  likely  that  in  tuberculosis  a  general 
hypersensitivenesH  is  developed,  and  may  be  the  underlying 
phenomenon.  These  reactions  must  therefore  be  considered  in 
the  light  of  what  will  be  set  forth  on  the  subject  of  hypersen- 
sitiveness  in  the  chapter  on  Immunity. 

The  Use  of  Old  Tuberculin  in  the  Diagnosis  of  Tuberculosis  in  Cattle. — 
111  cattle,  tuberculosis  may  be  present  without  giving  rise  to  apparent 
symptoms.  It  is  thus  important  from  the  point  of  view  of  human 
infection  that  an  early  diagnosis  should  be  made.  The  method  is 
applied  as  follows : — The  animals  are  kept  twenty-four  hours  in  their 
stalls,  and  the  temperature  is  taken  every  three  hours,  from  four  hours 
before  the  injection  till  twenty-four  a'fter.  The  average  temperature  in 
cattle  is  102'2°  F.  ;  30  to  40  centigrammes  of  tuberculin  are  injected,  and 
if  the  animal  be  tubercular  the  temperature  rises  2°  or  3°  F.  in  eight  to 
twelve  hours,  and  continues  elevated  for  ten  to  twelve  hours.  Bang,  who 
has  worked  most  at  the  subject,  lays  down  the  principle  that  the  more 
nearly  the  temperature  approaches  104°  F.  the  more  reason  for  suspicion 
is  there.  He  gives  a  record  of  280  cases  where  the  value  of  the  method 
was  tested  by  subsequent  post-mortem  examination.  He  found  that  with 
proper  precautions  the  error  was  only  3'3  per  cent.  The  method  has 
l>ti  11  largely  practised  in  all  parts  of  the  world,  and  is  of  great  value. 


288  TUBERCULOSIS 

(2)  Immunity  Phenomena  in  Tuberculosis. — Koch's  Tuber- 
culin-R. The  study  of  immunity  phenomena  in  tuberculosis 
dates  from  the  introduction  by  Koch  in  1897  of  the  substance 
denominated  by  him  "Tuberculin-R."  Koch's  new  researches 
consisted  (1)  of  an  attempt  to  immunise  animals  against  the 
tubercle  bacillus  by  employing  its  intracellular  toxins ;  (2)  of 
trying  to  utilise  such  an  immunisation  to  aid  the  tissues  of  an 
animal  already  attacked  with  tubercle  the  better  to  combat  the 
effects  of  the  bacilli.  The  method  of  obtaining  the  intracellular 
toxins  was  as  follows  :  Bacilli  from  young  virulent  cultures  were 
dried  in  vacuo,  and  disintegrated  in  an  agate  mill,  treated  with 
distilled  water  and  centrifugalised.  The  clear  fluid  was  decanted, 
and  is  called  by  Koch  "Tuberculin-O."  The  remaining  deposit 
was  again  dried,  ground,  treated  with  water  and  centrifugalised, 
the  clear  fluid  being  again  decanted,  and  this  process  was 
repeated  with  successive  residues  till  no  residue  remained. 
These  fluids  put  together  constitute  the  "  Tuberculin-R."  From 
the  fact  that  tuberculin-O  gave  no  cloudiness  when  glycerin  was 
added,  Koch  concluded  that  it  contained  the  substances  present 
in  the  glycerin-bouillon  extracts  originaUy  used  by  him,  and  he 
held  this  was  borne  out  by  the  readiness  with  which  a  tuberculin 
reaction  could  be  caused  by  it.  Similarly,  as  tuberculin-R  gave 
a  cloudiness  with  glycerin  and  did  not  readily  originate  a 
reaction,  he  considered  that  it  contained  different  products  of 
the  bacillus.  When  injected  into  animals  in  repeated  and 
increasing  doses,  -^  J^  mgrm.  being  the  initial  dose,  tuberculin-R 
was  said  to  produce  immunity  against  the  original  extract, 
against  tuberculin-O,  and  against  living  and  virulent  tubercle 
bacilli.  Further  research  has  not  confirmed  this  last  result. 

Itoch's  New  Tuberculin. — Another  preparation  has  also  been 
introduced,  known  as  "  Koch's  new  tuberculin "  (Bazillenemul- 
sion).  This  is  an  emulsion  of  ground  tubercle  bacilli  in  water 
containing  50  per  cent,  of  glycerin ;  it  thus  really  contains  both 
tuberculin-O  and  tuberculin-R. 

Scientific  enquiry  into  the  action  of  these  new  tuberculin 
preparations  has  resulted  in  attempts  being  made  to  recognise  in 
their  effects  phenomena  similar  to  those  produced  by  organisms 
such  as  the  typhoid  and  cholera  bacteria,  the  investigation  of 
which  has  brought  out  the  complex  processes  at  work  in  the 
reaction  of  an  organism  against  invading  bacteria.  The  phen- 
omena manifested  in  such  cases  consist  in  the  formation  of 
immune-bodies,  precipitins,  agglutinins,  and  opsonins. 

(1)  Immune-bodies  and  Precipitins.  —  Evidence  for  the 
existence  of  these  in  tuberculosis  has  been  sought  by  applying 


IMMTXITV    I'HKXOMKXA    IX  TriJKIK'ULO-TS     -JSO 

the  method  of  complement  fixation  (see  p.  130),  e.y.  the  serum 
of  a  tubercular  animal  being  mixed  with  tuberculin,  the  mixture 
is  tested  for  its  capacity  of  absorbing  complement.  Following 
this  line,  Wasserman  and  others  have  found  evidence  of  the 
presence  of  an  antituberculin  in  tubercular  foci,  and  this  is 
taken  as  an  indication  of  the  occurrence  of  a  vital  reaction 
against  the  poisons  of  an  invading  organism.  Generally  speak- 
ing, such  an  antituberculin  is  absent  from  the  blood  serum  of 
most  tubercular  patients.  It  is  present,  however,  in  the  serum 
of  such  individuals  after  they  have  been  subjected  to  repeated 
tuberculin  injections.  Here  it  is  chiefly  seen  when  a  patient  is 
losing  the  capacity  for  reacting  to  the  injections.  Another 
immunity  phenomenon  which  may  be  observed  is  the  formation 
of  a  precipitate  when  some  of  the  serum  of  a  tuberculous  patient 
is  added  to  a  solution  of  tuberculin,  the  mixture  being  allowed 
to  stand  for  twenty-four  hours  (precipitin  reaction).  The  exact 
relationship  of  such  precipitins  to  immune-bodies  is  still  doubtful ; 
that  it  is  a  close  one  is  shown  by  the  fact  that  such  precipitates 
have  the  property  of  absorbing  complement.  At  present  it  is 
enough  to  say  that  there  is  evidence  in  tubercular  infection  of  a 
vital  reaction  resulting  in  the  formation  of  antagonistic  bodies, 
which  may  include  both  immune-bodies  and  precipitins.  In  sup- 
port of  the  view  that  immune-bodies  exist  against  the  tubercle 
bacillus,  it  may  be  said  that  the  sera  of  certain  animals,  e.g. 
rabbit  and  ox,  when  mixed  with  tuberculin,  become  capable  of 
deviating  complement  from  a  haemolytic  combination. 

(2)  Agglutinins. — The  serum  of  tubercular  patients  has  been 
found  to  exert  an  agglutinating  action  on  the  tubercle  bacillus. 
A  convenient  method  is  to  add  different  amounts  of  serum,  com- 
mencing with,  say,  1  c.c.,  to  quantities  of  a  dilution  of  the  new 
tuberculin    (Bazillenemuhion)    equivalent    to     1     part   of   the 
bacterial  bodies  to  10,000  of  diluent,  and  leave  the  mixture  for 
twenty-four  hours  before  observing.     As  with  other  agglutinative 
observations,  it  is  difficult  to  correlate  the  degree  of  agglutinating 
power  of  the  serum  with  the  degree  of  immunisation  possessed 
1  > v  the  individual  from  which  it  was  derived.     The  method  has 
been  used  by  some  as  a  means  of  diagnosis,  but  its  value  is 
doubtful  and  is  certainly  inferior  to  the  methods  depending  on 
supersensitivcness. 

(3)  Opsonins. — The  serum  of  most  normal  men  and  of  several 
s|n'cies  of  animals  normally  contains  opsonins  to  the  tubercle 
bacillus.       The    opsonic   effect    is    also   manifested   in    varying 
degree  by  the  serum  during  the    course  of  natural  infection ; 
such  variations  are  considered  l>elow. 

19 


290  TUBERCULOSIS 

In  considering  the  relationships  of  the  specific  immune 
reactions  against  the  tubercle  bacillus,  it  is  to  be  noted  that 
while  the  existence  of  such  reactions  has  been  established,  the 
development  of  these  to  an  extent  likely  to  benefit  an  infected 
animal  is  limited,  and  the  production  of  such  a  lasting  immunity 
as  would  enable  it  to  resist  an  infection  or  to  throw  off  an 
infection  already  established  is  extremely  difficult  or  impossible. 
There  are  probably  factors  in  the  pathology  of  the  tubercular 
process  which  militate  against  such  an  occurrence.  This  pro- 
cess seems  to  differ  from  what  occurs  in  more  acute  infections,  in 
that  a  local  lesion  may  be  in  existence  for  a  very  considerable 
period  without  other  parts  of  the  body  being  much  or  at  all 
concerned.  This  is  especially  marked  in  certain  tubercular 
manifestations,  the  outstanding  example  of  which  is  lupus,  in 
which  for  years,  while  the  bacilli  are  present  and  active  in  the 
skin,  even  the  adjacent  lymphatic  glands  may  show  no  signs  of 
disease.  What  underlies  this  apparent  independence  of  the 
body  generally  in  relation  to  a  serious  condition  affecting  one 
locality  is  unknown.  Other  examples  of  a  similar  process  are 
found  in  leprosy  and  also  in  certain  chronic  suppurations  of  the 
skin.  f 

Therapeutic  Applications  of  the  Tuberculins. — We  have 
already  stated  that  the  use  of  the  old  tuberculin  to  mechanically 
remove  local  foci  of  tuberculosis  through  the  use  of  large  doses 
of  the  reagent  was  soon  found  to  be  impracticable,  but  both  this 
preparation  and  its  modifications  have  been  largely  used  in 
what  is  now  denominated  "vaccine-therapy."  It  has  been 
already  pointed  out  that  the  tubercular  process  is  peculiar  in 
that  the  disease  may  exist  locally  without  much  affecting  the 
general  health  of  the  infected  individual.  The  principle  of 
vaccine-therapy  may  roughly  be  said  to  be  to  bring  into  play  the 
potential  but  latent  defensive  mechanisms  of  the  body  with  the 
object  of  so  reinforcing  the  cells  locally  attacked  as  to  enable 
them  to  destroy  the  invading  bacteria.  This  is  effected  by 
introducing  into  the  body  small  doses  of  the  infecting  agent,  and 
is  in  reality  an  immunisation  carried  through  after  infection  has 
already  taken  place.  For  this  purpose  all  the  tuberculin  pre- 
parations, but  especially  tuberculin-R  and  the  "new  tuberculin," 
have  been  used.  In  the  case  of  both  the  latter,  doses  commen- 
cing with  from  TJ^  to  3^  mgrm.,  gradually  increased,  were 
given  every  second  day,  and  the  rule  originally  laid  down  for 
the  regulation  of  the  dosage  was  that  no  amount  should  be 
given  which  raised  the  temperature  more  than  '5°  F.  Opinion 
varied  as  to  the  efficacy  of  such  treatment.  There  was  little 


OPSONINS  IN  TUBERCULOSIS  291 

doubt  that  in  certain  cases  of  local  conditions,  such  as  lupus, 
tubercular  joints,  glands,  and  genito-urinary  tuberculosis,  improve- 
ment followed  its  application  ;  but  where  febrile  conditions 
indicated  that  general  disturbances  were  in  existence,  there  was 
little  justification  for  its  being  applied,  and  even  in  many  local 
conditions  the  absence  of  benefit  was  so  marked  that  by  many 
physicians  the  method  had  been  abandoned. 

Active  Immunisation  associated  with  Opsonic  Observations. — 
The  credit  of  rehabilitating  the  vaccine-therapy  of  tuberculosis 
and  of  defining  its  scope  belongs  to  Wright,  who  directed  atten- 
tion to  the  possibility  of  controlling  the  use  of  the  tuberculin  by 
observations  of  its  effect  on  the  opsonic  qualities  of  the  serum. 
Early  in  his  work  he  showed  that  tubercle  bacilli  when  sensitised 
by  an  appropriate  serum,  were  readily  phagocyted  by  the  poly- 
morpho-nucleate  leucocytes,  and  the  relative  sensitising  capacities 
of  serum  from  tubercular  and  non-tubercular  cases  has  been 
widely  studied.  According  to  Wright,  in  strictly  localised  tuber- 
culosis, the  opsonic  index  is  persistently  low,  varying  from  '1  to  '9, 
while  in  tuberculosis  with  general  disturbances  it  fluctuates 
greatly  from  day  to  day,  being  sometimes  below,  sometimes 
above  unity.  To  take  the  former  and  simpler  case,  he  holds 
that  if  the  treatment  with  injections  of  tuberculin  be  controlled 
by  noting  the  effect  produced  on  the  opsonic  index,  great 
improvement  in  the  patient's  condition  may  result.  Wright's 
interpretation  of  what  occurs  is  briefly  as  follows :  For 
reasons  unknown  the  opsonic  qualities  of  the  body  fluids 
may  become  abnormally  low,  and  the  tubercle  bacilli,  if 
they  gain  admission  to  the  body,  can  multiply  locally.  This 
multiplication  is  associated  with  a  still  further  local  diminution 
of  the  opsonins.  By  the  introduction  of  such  a  substance 
as  tuberculin,  the  bodily  mechanism,  whatever  it  is,  which 
produces  the  opsonins  is  stimulated,  and  a  rise  in  the  general 
opsonic  index  occurs.  Naturally  this  is  accompanied  by  a 
passing  to  the  site  of  infection  of  fluids  more  rich  in  opsonins 
than  previously,  the  activity  of  the  phagocytes  comes  into  play, 
and  the  tubercle  bacilli  are  destroyed.  But  any  such  vaccination 
process  must  be  controlled  by  constant  observations  of  the 
opsonic  index,  and  it  is  only  by  this  means,  not  only  that  good 
results  can  be  obtained,  but  that  the  production  of  harmful 
effects  can  be  prevented.  The  reason  of  this  is  that  in  a  great 
many  cases  the  injection  of  a  bacterial  vaccine  is  followed  by  a 
decrease  in  the  opsonic  qualities  of  the  serum, — the  occurrence 
of  a  negative  phase.  During  such  a  period  of  depression  there 
is  probably  an  increased  susceptibility  to  the  action  of  the 


292  TUBERCULOSIS 

bacilli.  Now,  in  order  to  get  permanent  benefit  from  the  vac- 
cination process,  repeated  injections  of  the  tuberculin  must  be 
practised,  and  if  an  injection  be  given  during  a  negative  phase, 
actual  harm  may  be  done.  The  course  of  a  successful  vaccina- 
tion is  that,  after  the  passing  off  of  the  negative  phase,  the 
opsonic  index  should  rise  to  above  its  original  level, — the 
occurrence  of  a  positive  phase.  It  is  when  this  positive  phase 
is  fully  developed  that  a  fresh  inoculation  can  be  practised  with 
success.  The  new  negative  phase  which  will  now  occur  may  not 
cause  a  drop  to  below  the  level  of  the  original  state  of  the  serum, 
and  the  hope  is  that  its  succeeding  positive  phase  will  carry  the 
opsonic  index  still  higher  and  ensure  a  still  greater  resistance 
to  the  bacterium.  There  are  very  great  variations  in  the  capa- 
cities shown  by  tubercular  patients  to  react  to  a  vaccination 
process.  In  certain  cases  good  positive  phases  are  readily  and 
quickly  produced,  while  in  others,  after  an  inoculation  the  negative 
phase  is  long  continued  and  may  even  show  no  tendency  to  pass 
into  a  positive  phase.  The  irregularities  in  the  opsonic  index  in 
cases  where  there  is  a  general  disturbance  of  metabolism,  Wright 
explains  by  supposing  that  they  result  from  very  irregular  auto- 
infections  of  the  patient's  body  by  tubercular  products  from  the 
local  lesions, — positive  and  negative  phases  being  produced  with- 
out the  purposive  quality  which  ought  to  characterise  a  success- 
ful therapeutic  vaccination.  Such  auto-infections  may  come 
about  in  various  ways,  and  Wright  is  of  opinion  that  exercise, 
for  instance,  may  disseminate  both  tubercular  products  and 
tubercle  bacilli, — he  having  noticed  in  certain  patients  a  fall  in 
the  opsonic  index  after  muscular  exertion.  For  ordinary  cases 
with  low  opsonic  index  and  no  evidence  of  constitutional  dis- 
turbance, an  amount  of  tuberculin  corresponding  to  from  one- 
thousandth  to  a  six-hundredth  of  a  milligramme  of  tubercle 
powder  is  a  sufficient  dose,  and  if  any  dose  seems  to  produce  a 
pronounced  negative  phase,  then  a  smaller  dose  ought  to  be  tried 
at  the  next  inoculation.  For  cases  clinically  tubercular,  where 
the  index  is  about  normal,  then  smaller  doses,  say,  the  equivalent 
of  a  two-thousandth  of  a  milligramme  or  less,  ought  to  be  used, 
— the  effect  on  the  index  being  carefully  watched.  In  any  case, 
the  dose  which  is  found  to  give  the  highest  positive  phase  is  the 
optimum  dose,  and  one  which  need  not  necessarily  be  increased. 
Cases  where  there  is  constitutional  disturbance  should  be  as  a 
rule  left  untreated. 

With  regard  to  the  results  obtained,  many  cases  have  been 
brought  forward  by  Wright  and  others  where  benefit  has 
followed  the  putting  into  practice  of  the  principles  enunciated, 


OPSONINS  IN  TUBERCULOSIS  293 

and  there  is  little  doubt  that  the  work  done  has  given  a  fresh 
start  to  the  active  immunisation  method  in  the  treatment  of 
tuberculosis.  An  outstanding  event  of  Wright's  work  in  this 
field  has  been  his  insistance  on  the  good  effects  produced  by 
extremely  small  doses  of  tuberculin  (down  to  the  four-thousandth 
of  a  milligramme)  given  at  fairly  long  intervals  (say  ten  days  or 
more).  With  regard  to  the  efficacy  of  the  opsonic  method  as 
affording  an  index  to  the  progress  of  a  case,  it  must  be  recognised 
that  the  method  is  still  on  its  trial,  and  it  is  doubtful  if  even  in 
the  work  of  the  most  careful  observers  the  limits  of  the  experi- 
mental error  of  the  opsonic  method  have  been  sufficiently  defined. 

Great  controversy  has  taken  place  as  to  whether  it  is  justi- 
fiable, in  the  treatment  of  tubercular  cases  with  tuberculin, 
merely  to  rely  on  the  observation  of  the  clinical  effects,  with- 
out having  recourse  to  the  constant  estimation  of  the  opsonic 
index,  which  Wright  considers  advisable.  There  is  no  doubt 
that  in  all  complicated  cases  of  tuberculosis,  such  as  lung 
affections  and  cases  of  multiple  foci  in  the  body,  the  treatment 
ought  to  be  in  the  hands  of  an  expert.  In  cases  of  strictly 
localised  tubercle,  however,  such  as  adenitis,  arthritis,  cystitis, 
or  lupus,  Wright  admits  that  in  many  cases,  without  much 
risk,  an  uncontrolled  treatment  may  be  undertaken.  The 
injections  ought  to  begin  with  doses  of  one-twenty-thousandth 
of  a  milligramme,  with  ten-day  intervals  intervening  between 
each  dose.  If  clinical  improvement  occurs,  the  dose  may  be 
gradually  increased  until  it-  reaches  one  four-thousandth  of  a 
milligramme  after  six  months.  If  the  treatment  of  any  other 
form  of  tuberculosis  be  undertaken  along  similar  lines,  the  pre- 
liminary injection  should  not  consist  of  more  than  one-fifty- 
thousandth  of  a  milligramme. 

The  whole  question  of  the  immunisation  treatment  of  tuber- 
culosis presents  many  difficulties,  and  it  is  the  merit  of  Wright's 
work  that  it  has  shed  fresh  light  on  some  of  these.  One  great 
difficulty  arises  from  the  great  chronicity  of  the  results  of  the 
infection  in  the  majority  of  human  cases.  It  is  probably  true 
not  only  of  man  but  of  many  species  of  animals  used  in  experi- 
mental inquiries,  that  many  individuals  are  on  the  border-line 
between  immunity  and  susceptibility.  From  the  widespread 
distribution  of  the  bacilli  in  centres  of  human  population,  it  is 
certain  that  the  opportunity  for  infection  arises  in  a  very  large 
proportion  of  the  race ;  in  many  cases  no  results  follow  infection, 
and  in  many  others  small  lesions  occur  which  do  not  develop 
further ;  this  has  actually  been  shown  by  morbid  anatomists  to  be 
the  case.  The  disease  being  thus  so  often  characterised  by  transient 


294  TUBERCULOSIS 

local  effects  without  constitutional  disturbance,  the  course  of  an 
immunisation  may  be  expected  to  be  rather  different  from  that 
obtaining  in  an  ordinary  acute  affection,  though  the  underlying 
processes  may  be  of  the  same  nature.  It  is  difficult,  for  instance, 
on  account  of  the  slowness  of  tubercular  processes,  to  define 
recovery  from  an  attack  of  the  disease,  or  to  speak  of  an  animal 
recovering  from  the  effect  of  an  inoculation  during  an  immunisa- 
tion. It  follows  that  little  is  known  regarding  an  attenuation  of 
the  tubercle  bacillus  analogous  to  what  is  an  important  feature 
in  immunisations  against  other  organisms. 

Antitubercular  Sera. — Several  attempts  have  been  made  to 
treat  tuberculosis  with  the  serum  of  animals  immunised  by  the 
tubercle  bacillus  or  its  products.  The  most  successful  is  perhaps 
that  of  Maragliano.  This  author  distinguishes  between  the  toxic 
materials  contained  in  the  bodies  of  the  bacilli  (which  withstand, 
unchanged,  a  temperature  of  100°  C.)  and  those  secreted  into 
the  culture  fluid  (which  are  destroyed  by  heat).  The  substance 
used  by  him  for  immunising  his  animals  consists  of  three  parts 
of  the  former  and  one  of  the  latter.  The  animals  employed  are 
the  dog,  the  ass,  the  horse.  The  serum  obtained  from  these  is 
capable  of  protecting  healthy  animals  against  an  otherwise  fatal 
dose  of  tuberculin,  but  very  little  importance  can  be  attached  to 
this  result.  Maragliano  does  not  appear  to  have  studied  the 
effects  of  this  serum  on  tubercular  animals,  but  it  has  been  tried 
in  a  great  number  of  cases  of  human  tuberculosis,  2  c.c.  being 
injected  subcutaneously  every  two  days.  Improvement  is  said 
to  have  taken  place  in  a  certain  proportion,  especially  of  mild 
non-febrile  cases. 

An  antitubercular  serum  has  also  been  introduced  by  Marmorek. 
This  observer  considers  that  the  tubercle  bacillus  cannot  produce 
in  ordinary  media  the  toxins  which  it  originates  when  exposed 
to  the  antagonism  of  the  bodily  cells.  He  tries  to  make  good 
this  defect  by  first  growing  it  in  a  serum  antagonistic  to  some  of 
the  phagocytic  cells  of  the  body ;  for  this  a  leucotoxic  serum  is 
used.  When  the  bacillus  has  grown  in  this  presumably  favour- 
able soil,  it  is  transferred  to  a  medium  containing  a  substance 
which  may  be  unfavourable ;  and  for  this  there  is  employed  a 
medium  containing  liver  extract,  the  liver  being  an  organ  in 
which  in  man  tubercular  lesions  are  comparatively  rare.  The 
bacilli  being  thus  accustomed  to  an  unfavourable  surrounding 
are  used  for  immunising  animals,  the  serum  of  which  is  now 
suitable  for  the  treatment  of  human  tuberculosis.  There  is  con- 
siderable diversity  of  opinion  as  to  the  efficacy  of  Marmorek's 
serum  as  a  therapeutic  agent. 


METHODS  OF  EXAMINATION  -   295 

Methods  of  Examination. — (1)  Microscopic  Examinatioii. 
Tuberculosis  is  one  of  the  comparatively  few  diseases  in  which 
a  diagnosis  can  usually  be  definitely  made  by  microscopic 
examination  alone.  In  the  case  of  sputum,  one  of  the  yellowish 
fragments  which  are  often  present  ought  to  be  selected ;  dried 
films  are  then  prepared  in  the  usual  way,  and  stained  by  the 
Ziehl-Neelsen  stain  (p.  108).  In  the  case  of  urine  or  other  fluids, 
a  deposit  should  first  be  obtained  by  centrifugalising  a  quantity 
in  a  test-tube,  or  by  allowing  the  fluids  to  stand  in  a  tall  glass 
vessel  (an  ordinary  burette  is  very  convenient).  Film  prepara- 
tions are  then  made  with  the  deposit  and  treated  as  before.  If 
a  negative  result  is  obtained  in  a  suspected  case,  repeated  exam- 
ination should  be  undertaken.  To  avoid  risk  of  contamination 
with  the  smegma  bacillus,  the  meatus  of  the  urethra  should  be 
cleansed  and  the  urine  first  passed  should  be  rejected,  or  the 
urine  may  be  drawn  off  with  a  sterile  catheter.  As  stated  above, 
it  is  only  exceptionally  that  difficulty  will  arise  to  the  experienced 
observer  from  this  cause.  (For  points  to  be  attended  to,  vide  p. 
280).  The  detection  of  tubercle  bacilli  by  microscopical  methods 
in  sputum,  pus,  faeces,  and  even  tissues,  has  been  greatly  facilitated 
by  the  recent  introduction  of  a  preparation  called  "antiformin." 
This  is  a  mixture  of  equal  parts  of  liquor  soda3  chlorinatae  (B.P.) 
and  of  a  15  per  cent,  solution  of  caustic  soda.  It  has  a  re- 
markable disintegrative  and  dissolving  action  on  the  tissues,  etc., 
so  that  after  it  has  been  allowed  to  act  on  sputum,  for  example, 
and  the  mixture  is  centrifugalised,  the  resulting  deposit  is  scanty 
and  the  tubercle  bacilli,  if  present,  are  accordingly  greatly 
concentrated.  The  time  necessary  may  be  judged  of  by  the 
appearance  of  the  mixture,  but  it  will  generally  be  found  that 
the  desired  result  will  be  obtained  if  one  part  of  antiformin  be 
added  to  five  or  six  parts  of  sputum  and  allowed  to  act  for  two  or 
three  hours. 

(2)  Inoculation. — The  guinea-pig  is  the  most  suitable  animal. 
If  the  material  to  be  tested  is  a  fluid,  it  is  injected  subcutaneously 
or  into  the  peritoneum ;  if  solid  or  semi-solid,  it  is  placed  in  a 
small  pocket  in  the  skin,  or  it  may  be '  thoroughly  broken  up 
in  sterile  water  or  other  fluid  and  the  emulsion  injected.     By 
this  method,  material  in  which  no  tubercle  bacilli  can  be  found 
microscopically  may  sometimes  be  shown  to  be  tubercular. 

(3)  Cultivation. — Owing   to   the   difficulties   this   is   usually 
quite   impracticable    as   a   means  of   diagnosis,    and    it   is    also 
unnecessary.     The    best    method  to   obtain    pure    cultures    is 
to  produce   tuberculosis   in   a   guinea-pig   by   inoculation  with 
tubercular  material,  and  then,  killing  the  animal  after  four  or 


296   '  TUBERCULOSIS 

five  weeks,  to  inoculate  tubes  of  solidified  blood  serum,  under 
strict  aseptic  precautions,  with  portions  of  a  tubercular  organ, 
e.g.  the  spleen.  The  portions  of  tissue  should  be  fairly  large, 
and  should  be  well  rubbed  into  the  broken  surface  of  the  medium. 
Cultures  may,  however,  be  obtained  from  sputum  by  means  of 
antiformin,  as  this  substance  readily  kills  most  of  the  ordinary 
bacteria  and  has  comparatively  slight  effect  on  the  tubercle 
bacillus.  Antiforrain  should  be  allowed  to  act  on  sputum  in 
the  proportion  and  for  the  time  mentioned  in  paragraph  (1),  the 
mixture  should  then  be  centrifugalised,  the  supernatant  fluid 
removed,  and  the  deposit  washed  with  sterile  water  and  again 
centrifugalised,  these  processes  being  repeated  several  times.  If, 
then,  inoculations  be  made  from  the  deposit  on  blood  serum  or 
on  Dorset's  egg  medium,  pure  cultures  of  the  tubercle  bacillus 
may,  in  some  instances,  be  obtained.  The  method  is  one  which 
gives  good  results.  Another  somewhat  similar  method  is  that 
introduced  by  Twort ;  in  this,  portions  of  sputum  are  exposed 
to  the  action  of  a  2  per  cent,  solution  of  ericolin  (a  glucoside)  for 
an  hour  at  38°  C.,  and  thereafter  cultures  are  made  on  Dorset's 
medium. 

(4)  Reactive  phenomena. — The  presence  of  immune-substances 
in  the  blood  and  the  tuberculin  reaction,  along  with  the  methods 
of  applying  the  respective  tests,  have  been  described  above 
(p.  284). 


CHAPTER   XL 

LEPROSY. 

LEPROSY  is  a  disease  of  great  interest,  alike  in  its  clinical  and 
pathological  aspects;  whilst  from  the  bacteriological  point  of 
view,  also,  it  presents  some  striking  peculiarities.  The  invariable 
association  of  large  numbers  of  characteristic  bacilli  with  all 
leprous  lesions  is  a  well-established  fact,  and  yet,  so  far, 
attempts  to  cultivate  the  bacilli  outside  the  body,  or  to  produce 
the  disease  experimentally  in  animals,  have  been  attended  with 
failure.  Leprosy,  so  far  as  is  known,  is  a  disease  which  is  con- 
fined to  the  human  subject,  but  it  has  a  very  wide  geographical 
distribution.  It  occurs  in  certain  parts  of  Europe — Norway, 
Russia,  Greece,  etc.,  but  is  commonest  in  Asia,  occurring  in 
Syria,  Persia,  etc.  It  is  prevalent  in  Africa,  being  especially 
found  along  the  coast,  in  the  Pacific  Islands,  in  the  warmer 
parts  of  North  and  South  America,  and  also  to  a  small  extent 
in  the  northern  part  of  North  America.  In  all  these  various 
regions  the  disease  presents  the  same  general  features,  and  the 
•  study  of  its  pathological  and  bacteriological  characters,  wherever 
such  has  been  carried  on,  has  yielded  similar  results. 

Pathological  Changes. — Leprosy  is  characteristically  a  chronic 
disease,  in  which  there  is  a  great  amount-  of  tissue  change,  with 
comparatively  little  necessary  impairment  of  the  general  health. 
In  other  words,  the  local  effects  of  the  bacilli  are  well  marked, 
often  extreme,  whilst  the  toxic  phenomena  are  proportionately 
at  a  minimum. 

There  are  two  chief  forms  of  leprosy.  The  one,  usually  called 
the  tubercular  form, — lepra  tuberosa  or  tulerculosa, — is  character- 
ised by  the  growth  of  granulation  tissue  in  a  nodular  form  or 
as  a  diffuse  infiltration  in  the  skin,  in  mucous  membranes,  etc., 
great  disfigurement  often  resulting.  In  the  other  form,  the 
anaesthetic, — maculo-anajsthetic  of  Hansen  and  Looft, — the  out- 
standing changes  are  in  the  nerves,  with  consequent  anaesthesia, 
paralysis  of  muscles,  and  trophic  disturbances. 

297 


298 


LEPROSY 


In  the  tubercular  form,  the  disease  usually  starts  with  the 
appearance  of  erythematous  patches  attended  by  a  small  amount 
of  fever,  and  these  are  followed  by  the  development  of  small 
nodular  thickenings  in  the  skin,  especially  of  the  face,  of  the 
backs  of  hands  and  feet,  and  of  the  extensor  aspects  of  arms  and 
legs.  These  nodules  enlarge  and  produce  great  distortion  of  the 
surface,  so  that,  in  the  case  of  the  face,  an  appearance  is  produced 


FIG.  86. — Sections  through  leprous  skin,  showing  the  masses  of 
cellular  granulation  tissue  in  the  cutis  ;  the  dark  points  are  cells 
containing  bacilli  deeply  stained. 

Paraffin  section  ;  Ziehl-Neelsen  stain,      x  80. 


which  has  been  described  as  "  leonine."  The  thickenings  occur 
chiefly  in  the  cutis  (Fig.  86),  to  a  less  extent  in  the  subcutaneous 
tissue.  The  epithelium  often  becomes  stretched  over  them, 
and  an  oozing  surface  becomes  developed,  or  actual  ulceration 
may  occur.  The  cornea  and  other  parts  of  the  eye,  the  mucous 
membrane  of  the  mouth,  larynx,  and  pharynx,  may  be  the  seat 
of  similar  nodular  growths.  Internal  organs,  especially  the 
spleen,  liver,  and  testicles,  may  become  secondarily  affected.  In 
all  situations  the  change  is  of  the  same  nature,  consisting  in  an 


BACILLUS  OF  LEPROSY  299 

abundant  formation  of  granulation  tissue,  nodular  or  diffuse  in 
its  arrangement.  In  this  tissue  a  large  proportion  of  the  cells 
are  of  rounded  or  oval  shape,  like  hyaline  leucocytes ;  a  number 
of  these  may  be  of  comparatively  large  size,  and  may  show 
vacuolation  of  their  protoplasm  and  a  vesicular  type  of  nucleus. 
These  are  often  known  as  "  lepra-cells."  Amongst  the  cellular 
elements  there  is  a  varying  amount  of  stroma,  which  in  the 
earlier  lesions  is  scanty  and  delicate,  but  in  the  older  lesions 
may  be  very  dense.  Periarteritis  is  a  common  change,  and  very 
frequently  the  superficial  nerves  become  involved  in  the  nodules, 
and  undergo  atrophy.  The  tissue  in  the  leprous  lesions  is 
comparatively  vascular,  at  least  when  young,  and,  unlike 
tubercular  lesions,  never  shows  caseation.  Some  of  the  lepra 
cells  may  contain  several  nuclei,  but  we  do  not  meet  with  cells 
resembling  in  their  appearance  tubercle  giant-cells,  nor  does  an 
arrangement  like  that  in  tubercle  follicles  occur. 

In  the  anesthetic  form,  the  lesion  of  the  nerves  is  the  out- 
standing feature.  These  are  the  seat  of  diffuse  infiltrations, 
which  lead  to  the  destruction  of  the  nerve  fibres.  In  the  earlier 
stages,  in  which  the  chief  symptoms  are  pains  along  the  nerves, 
there  occur  patches  on  the  skin,  often  of  considerable  size,  the 
margins  of  which  show  a  somewhat  livid  congestion.  Later, 
these  patches  become  pale  in  the  central  parts,  and  the  periphery 
becomes  pigmented.  There  then  follow  remarkable  series  of 
trophic  disturbances,  in  which  the  skin,  muscles,  and  bones  are 
especially  involved.  The  skin  often  becomes  atrophied,  parch- 
ment-like, and  anaesthetic ;  frequently  pemphigoid  bulke  or  other 
skin  eruptions  occur.  Partly  owing  to  injury  to  which  the  feet 
and  arms  are  liable  from  their  anaesthetic  condition,  and  partly 
owing  to  trophic  disturbances,  necrosis  and  separation  of  parts 
are  liable  to  occur.  In  this  way  great  distortion  results.  The 
lesions  in  the  nerves  are  of  the  same  nature  as  those  described 
above,  but  the  granulation  tissue  is  scantier,  and  has  a  greater 
tendency  to  undergo  cicatricial  contraction.  This  is  to  be 
associated  with  the  fact  that  the  bacilli  are  present  in  fewer 
numbers. 

Bacillus  of  Leprosy. — This  bacillus  was  first  observed  in 
leprous  tissues  by  Hansen  in  1871,  and  was  the  subject  of  several 
communications  by  him  in  1874  and  later.  Further  researches, 
first  by  Neisser  in  1879,  and  afterwards  by  observers  in 
various  parts  of  the  world,  agreed  in  their  main  results,  and 
confirmed  the  accuracy  of  Hansen's  observations.  The  bacilli,  as 
seen  in  scrapings  of  ulcerated  leprous  nodules,  or  in  sections, 
have  the  following  characters  : — They  are  thin  rods  of  practically 


300  LEPROSY 

the  same  size  as  tubercle  bacilli,  which  they  also  resemble  both 
in  appearance  and  in  staining  reaction.  They  are  straight  or 
slightly  curved,  and  usually  occur  singly,  or  two  may  be  attached 
end  to  end ;  but  they  do  not  form  chains.  When  stained  they 
may  have  a  uniform  appearance,  or  the  protoplasm  may  be 
fragmented,  so  that  they  appear  like  short  rows  of  cocci.  They 
often  appear  tapered  at  one  or  both  extremities  ;  occasionally 


ffi 


FlG.  87. — Superficial  part  of  leprous  skin  ;  the  cells  of  the  granula- 
tion tissue  appear  as  dark  patches,  owing  to  the  deeply  stained  bacilli 
in  their  interior.  In  the  upper  part  a  process  of  epithelium  is  seen. 

Paraffin  section  ;  stained  with  carbol-fuchsin  and  Bismarck-brown, 
x  500. 

there  is  slight  club-like  swelling.  Degenerated  and  partially 
broken-down  forms  are  also  seen.  They  take  up  the  basic 
aniline  stains  more  readily  than  tubercle  bacilli,  but  in  order 
to  stain  them  deeply,  a  powerful  stain,  such  as  carbol-fuchsin, 
is  necessary.  When  stained,  they  strongly  resist  decolorising, 
though  they  are  more  easily  decolorised  than  tubercle  bacilli 
(p.  108).  The  bacilli  are  also  readily  stained  by  Gram's  method. 
Regarding  the  presence  of  spores,  practically  nothing  is  known, 
though  some  of  the  unstained  or  stained  points  may  be  of  this 


POSITION  OF  THE  BACILLI  301 

nature.     We  have,  however,  no  means  of  testing  their  powers  of 
resistance.     Leprosy  bacilli  are  non-motile. 

Position  of  the  Bacilli. — They  occur  in  enormous  numbers 
in  the  leprous  lesions,  especially  in  the  tubercular  form — in  fact, 
so  numerous  are  they  that  the  granulation  tissue  in  sections, 
properly  stained  as  above,  presents  quite  a  red  colour  under  a 
low  power  of  the  microscope  (Plate  II.,  Fig!  8).  The  bacilli 


FIG.  88. — High -power  view  of  portion  of  leprous  nodule,  showing  the 
arrangement  of  the  bacilli  within  the  cells  of  the  granulation  tissue. 

Paraffin  section  ;  stained  with  carbol-fuchsiu  and  methylene-blue 
x  1100. 

occur  for  the  most  part  within  the  protoplasm  of  the  round 
cells  of  the  granulation  tissue,  and  are  often  so  numerous  that 
the  structure  of  the  cells  is  quite  obscured  (Fig.  87).  They 
are  often  arranged  in  bundles  which  contain  several  bacilli 
lying  parallel  to  one  another,  though  the  bundles  lie  in  various 
directions  (Fig.  88  and  Plate  II.,  Fig.  9).  The  appearance  thus 
presented  by  the  cells  filled  with  bacilli  is  very  characteristic. 
Bacilli  are  also  found  free  in  the  lymphatic  spaces,  but  the  greater 
ii'imber  are  undoubtedly  contained  within  the  cells.  They  are 


302  LEPROSY 

also  found  in  spindle-shaped  connective-tissue  cells,  in  endothelial 
cells,  and  in  the  walls  of  blood  vessels.  They  are  for  the  most 
part  confined  to  the  connective  tissue,  but  a  few  may  be 
seen  in  the  hair  follicles  and  glands  of  the  skin.  Occasionally 
one  or  two  may  be  found  in  the  surface  epithelium,  where  they 
probably  have  been  carried  by  leucocytes,  but  this  position  is,  on 
the  whole,  exceptional.  They  also  occur  in  large  numbers  in  the 
lymphatic  glands  associated  with  the  affected  parts.  In  the 
internal  organs, — liver,  spleen,  etc., — when  leprous  lesions  are 
present,  the  bacilli  are  also  found,  though  in  relatively  smaller 
numbers.  In  the  nerves  in  the  anaesthetic  form  they  are  com- 
paratively few,  and  in  the  sclerosed  parts  it  may  be  impossible  to 
find  any.  There  are  few  also  in  the  skin  patches  referred  to 
above  as  occurring  in  this  form  of  the  disease. 

Their  spread  is  chiefly  by  the  lymphatics,  though  distribution 
by  the  blood  stream  also  occurs.  They  are  said  to  have  been 
found  in  the  blood  during  the  presence  of  fever  and  the  eruption 
of  fresh  nodules,  and  they  have  also  been  observed  in  the  blood 
vessels  post  mortem,  chiefly  contained  within  leucocytes.  Recent 
observations  (e.g.  those  of  Doutrelepont  and  Wolters)  show  that 
the  bacilli  may  be  more  widely  spread  throughout  the  body  than 
was  formerly  supposed.  A  few  may  be  detected  in  some  cases 
in  various  organs  which  show  no  structural  change,  especially  in 
the  capillaries.  The  brain  and  spinal  cord  are  almost  exempt, 
but  in  some  cases  bacilli  have  been  found  even  within  nerve 
cells. 

Eelations  to  the  Disease. — Attempts  to  obtain  pure  cultures 
of  the  leprosy  bacillus  have  so  far  been  unsuccessful.  Clegg  has 
recently  attempted  to  grow  the  organism  in  association  with 
amoebae  and  other  bacteria  on  agar  plates,  and  has  obtained  a 
short  acid-fast  bacillus  which  does  not  grow  on  ordinary  media, 
and  which  has  been  carried  through  several  generations  in  the 
conditions  mentioned.  The  identity  of  this  organism  with  the 
leprosy  bacillus  has,  however,  not  been  established.  Attempts 
to  transfer  the  disease  to  animals,  including  monkeys,  have  been 
unsuccessful.  When  a  small  portion  of  leprous  material  is 
transplanted,  a  nodule  may  result  in  which  leprosy  bacilli  may  be 
demonstrated  for  some  time,  but  this  probably  represents  merely 
the  reaction  to  a  foreign  body ;  there  is  no  sufficient  evidence 
that  the  bacilli  undergo  multiplication,  and  it  is  impossible  to 
continue  such  lesions  in  fresh  animals.  The  only  exception  to 
this  statement  is  afforded  by  the  experiments  of  Melcher  and 
Orthmann,  who  inoculated  the  anterior  chamber  of  the  eye  of 
rabbits  with  leprous  material,  the  inoculation  being  followed  by 


RELATIONS  TO  THE  DISEASE  303 

an  extensive  growth  of  nodules  in  the  lungs  and  internal  organs, 
which  they  affirmed  contained  leprosy  bacilli.  It  has  been 
questioned,  however,  by  several  authorities  whether  the  organisms 
in  the  nodules  were  really  leprosy  bacilli,  and  up  to  the  present 
we  cannot  say  that  there  is  any  satisfactory  proof  that  the 
disease  can  be  transmitted  to  any  of  the  lower  animals.  Diph- 
theroid  bacilli  of  more  than  one  variety  have  been  cultivated 
from  the  blood  and  tissues  of  leprous  patients  by  Babes  and 
others.  Their  presence  would  appear  to  be  by  no  means  in- 
frequent, but  it  is  not  possible  to  say  at  present  what  their 
significance  is. 

It  is  interesting  to  note  that  a  disease  occurs  under  natural 
conditions  in  rats  which  presents  many  points  of  close  similarity 
to  leprosy.  It  is  very  widespread,  having  been  observed  in 
Europe,  Asia,  America,  and  Australia ;  an  excellent  description 
has  been  given  by  Dean.  In  this  affection  there  are  lesions 
in  the  skin  which  resemble  those  in  leprosy,  and  the  cells  con- 
tain enormous  numbers  of  an  acid-fast  bacillus.  The  disease 
can  be  transmitted  to  rats  by  inoculation  with  the  tissue  juices 
containing  the  bacilli,  but  not  to  animals  of  other  species.  All 
attempts  to  cultivate  the  characteristic  organism  outside  the 
body  have  failed,  but  Dean  has  obtained  a  diphtheroid  bacillus 
— a  result  of  interest  in  relation  to  what  has  been  found  in 
leprosy.  Whether  this  disease  has  any  relation  to  leprosy  in  the 
human  subject  is  very  doubtful,  but  the  facts  which  have  been 
ascertained  may  prove  of  high  importance  in  connection  with 
the  pathology  of  the  latter  disease. 

It  would  also  appear  that  the  disease  is  not  readily  inoculable 
in  the  human  subject.  In  a  wrell-known  case  described  by  Arning, 
a  criminal  in  the  Sandwich  Islands  was  inoculated  in  several 
parts  of  the  body  with  leprosy  tissue.  Two  or  three  years  later, 
well-marked  tubercular  leprosy  appeared,  and  led  to  a  fatal  result. 
This  experiment,  however,  is  open  to  the  objection  that  the 
individual  before  inoculation  had  been  exposed  to  infection  in  a 
natural  way,  having  been  frequently  in  contact  with  lepers.  In 
other  cases,  inoculation  experiments  on  healthy  subjects  and 
inoculations  in  other  parts  of  leprous  individuals  have  given 
negative  results.  It  has  been  supposed  by  some  that  the  failure 
to  obtain  cultures  and  to  reproduce  the  disease  experimentally 
may  be  partly  due  to  the  bacilli  in  the  tissues  being  dead.  That 
many  of  the  leprous  bacilli  are  in  a  dead  condition  is  quite 
possible,  in  view  of  the  long  period  during  which  dead  tubercle 
bacilli  introduced  into  the  tissues  of  animals  retain  their  form 
and  staining  reaction.  There  is  also  the  fact  that  from  time  to 


304  LEPROSY 

time  in  leprous  subjects  there  occur  febrile  attacks,  which  are 
followed  by  a  fresh  outbreak  of  nodules,  and  it  would  appear 
that  especially  at  these  times  multiplication  of  the  bacilli  takes 
place  more  actively. 

The  facts  stated  with  regard  to  cultivation  and  inoculation 
experiments  go  to  distinguish  the  leprosy  bacillus  all  the  more 
strongly  from  other  organisms.  Some  have  supposed  that  leprosy 
is  a  form  of  tubercle,  or  tubercle  modified  in  some  way,  but  for 
this  there  appears  to  us  to  be  no  evidence.  It  should  also  be 
mentioned  that  tubercle  is  a  not  uncommon  complication  in 
leprous  subjects,  in  which  case  it  presents  the  ordinary  characters. 
It  has  been  found  that,  a  considerable  proportion  of  lepers  react 
to  tuberculin  like  tubercular  patients.  This  result  has  been 
variously  interpreted,  some  considering  that  tuberculosis  is  also 
present  in  such  cases,  whilst  others  maintain  that  the  reaction 
may  be  given  in  the  absence  of  tubercle.  If,  as  is  probable,  the 
latter  is  the  case,  the  result  most  likely  depends  on  the  close 
relationship  of  the  organisms  of  the  two  diseases ;  it  by  no  means 
proves  their  identity.  Another  curious  fact  is  that  the  Wasser- 
mann  reaction  (p.  131)  may  be  given  by  the  serum  of  leprous 
patients  (in  about  50  per  cent.,  according  to  some  observers) ; 
this  would  seem  to  be  quite  independent  of  the  concurrent 
presence  of  syphilis,  but  it  is  not  possible  at  present  to  give  an 
explanation  of  the  phenomenon. 

The  mode  by  which  leprosy  is  transmitted  has  been  the  subject 
of  great  controversy,  and  is  one  on  which  authorities  still  hold 
opposite  opinions.  Some  consider  that  it  is  a  hereditary  disease, 
or  at  least  that  it  is  transmitted  from  a  parent  to  the  offspring ; 
others,  again,  that  it  is  transmitted  by  direct  contact.  There 
appears  to  be  no  doubt,  however,  that  on  the  one  hand  leprous 
subjects  may  bear  children  free  from  leprosy,  and  that  on  the 
other  hand  healthy  individuals  entering  a  leprous  district  may 
contract  the  disease,  though  this  rarely  occurs.  Of  the  latter 
occurrence  there  is  the  well-known  instance  of  Father  Damien, 
who  contracted  leprosy  after  going  to  the  Sandwich  Islands.  In 
view  of  all  the  facts,  there  can  be  little  doubt  that  leprosy  in 
certain  conditions  may  be  transmitted  by  direct  contact,  though 
its  contagiousness  is  not  of  a  high  order. 

Methods  of  Diagnosis. — Film  preparations  should  be  made 
with  the  discharge  from  any  ulcerated  nodule  which  may  be 
present,  or  from  the  scraping  of  a  portion  of  excised  tissue,  and 
should  be  stained  as  above  described.  The  presence  of  large 
numbers  of  bacilli  situated  within  the  cells  and  giving  the  staining 
reaction  of  leprosy  bacilli,  is  conclusive.  It  is  more  satisfactory, 


METHODS  OF  DIAGNOSIS  305 

however,  to  make  microscopic  sections  through  a  portion  of  the 
excised  tissue,  when  the  structure  of  the  nodule  and  the  arrange- 
ment of  the  bacilli  can  be  readily  studied.  The  points  of  differ- 
ence between  leprosy  and  tubercle  have  already  been  stated,  and 
in  most  cases  there  is  really  no  difficulty  in  distinguishing  the 
two  conditions. 


20 


CHAPTER   XII. 

GLANDERS  AND  RHINOSCLEROMA. 

GLANDERS. 

THE  bacillus  of  glanders  (bacillus  mallei ;  Fr.,  bacille  de  la  morve ; 
Ger.,  Rotzbacillus)  was  discovered  by  Loffler  and  Schutz,  the 
announcement  of  this  discovery  being  made  towards  the  end 
of  1882.  They  not  only  obtained  pure  cultures  of  this  organism 
from  the  tissues  in  the  disease,  but  by  experiments  on  horses 
and  other  animals  conclusively  established  its  causal  relationship. 
These  have  been  fully  confirmed.  The  same  organism  has  also 
been  cultivated  from  the  disease  in  the  human  subject,  first  by 
Weichselbaum  in  1885,  who  obtained  it  from  the  pustules  in  a 
case  of  acute  glanders  in  a  woman,  and  by  inoculation  of  animals 
obtained  results  similar  to  those  of  Loffler  and  Schutz. 

Within  more  recent  times  a  substance,  mallein,  has  been 
obtained  from  the  cultures  of  the  glanders  bacillus  by  a  method 
similar  to  that  by  which  tuberculin  was  prepared,  and  has  been 
found  to  produce  effects  in  animals  suffering  from  glanders  corre- 
sponding to  those  produced  by  tuberculin  in  tuberculous  animals. 

The  Natural  Disease. — Glanders  chiefly  affects  the  equine 
species — horses,  mules,  and  asses.  Horned  cattle,  on  the  other 
hand,  are  quite  immune,  whilst  goats  and  sheep  occupy  an  inter- 
mediate position,  the  former  being  rather  more  susceptible  and 
occasionally  suffering  from  the  natural  disease.  It  also  occurs 
in  some  of  the  carnivora — cats,  lions  and  tigers  in  menageries, 
which  animals  are  infected  from  the  carcasses  of  animals  affected 
with  the  disease.  Many  of  the  small  rodents  are  highly  sus- 
ceptible to  inoculation  (vide  infra). 

Glanders  is  also  found  in  man  as  the  result  of  direct  inocula- 
tion on  some  wound  of  the  skin  or  other  part  by  means  of  the 
discharges  or  diseased  tissues  of  an  animal  affected,  and  hence  is 
commonest  amongst  grooms  and  others  whose  work  brings  them 
into  contact  with  horses ;  even  amongst  them  it  is  a  comparatively 
rare  disease. 

306 


THE  NATURAL  DISEASE  307 

In  horseS  the  lesions  are  of  two  types,  to  which  the  names  "glanders  " 
proper  and  "farcy"  have  been  given,  though  both  may  exist  together. 
In  glanders  proper,  the  septum  nasi  and  adjacent  parts  are  chiefly  atfected, 
there  occurring  in  the  mucous  membrane  nodules  which  are  at  first  firm 
and  of  somewhat  translucent  grey  appearance.  The  growth  of  these  is 
attended  usually  by  inflammatory  swelling  and  profuse  catarrhal  dis- 
charge. Afterwards  the  nodules  soften  in  the  centre,  break  down,  and 
give  rise  to  irregular  liberations.  Similar  lesions,  though  in  less  degree, 
may  be  found  in  the  respiratory  passages.  Associated  with  these  lesions 
there  is  usually  implication  of  the  lymphathic  glands  in  the  neck,  media- 
stinum, etc.  ;  and  there  may  be  in  the  lungs,  spleen,  liver,  etc.,  nodules 
of  the  size  of  a  pea  or  larger,  of  greyish  or  yellow  tint,  firm  or  somewhat 
softened  in  the  centre,  and  often  surrounded  by  a  congested  zone.  The 
term  "farcy"  is  applied  to  the  affection  of  the  superficial  lymphatic 
vessels  and  glands,  which  is  specially  seen  where  infection  takes  place 
through  an  abrasion  of  the  skin,  such  as  is  often  produced  by  the  rubbing 
of  the  harness.  The  lymphatic  vessels  become  irregularly  thickened,  so 
as  to  appear  like  knotted  cords,  and  the  associated  lymphatic  glands 
become  enlarged  and  firm,  though  suppurative  softening  usually  follows, 
and  there  may  be  ulceration.  These  thickenings  are  often  spoken  of  as 
"  farcy  buds  "  and  "  farcy  pipes."  In  farcy,  also,  secondary  nodules  may 
occur  in  internal  organs  and  the  nasal  mucous  membrane.  The  disease 
is  often  present  in  a  "  latent  form,"  and  its  presence  can  only  be  detected 
by  the  mallein  test  (vide  infra).  In  the  ass  the  disease  runs  a  more 
acute  course  than  in  the  horse. 

In  man  the  disease  is  met  with  in  two  forms,  an  acute  and  a 
chronic — though  intermediate  forms  also  occur,  and  chronic  cases 
may  take  on  the  characters  of  the  acute  disease.  The  site  of 
inoculation  is  usually  on  the  hand  or  arm, — by  means  of  some 
scratch  or  abrasion,  or  possibly  by  infection  along  a  hair  follicle, — 
sometimes  on  the  face,  and  occasionally  on  the  mucous  membrane 
of  the  mouth,  nose,  or  eye.  In  the  acute  form  there  appears  at 
the  site  of  inoculation  inflammatory  swelling,  attended  usually  with 
spreading  redness,  and  the  lymphatics  in  relation  to  the  part  also 
become  inflamed,  the  appearances  being  those  of  a  "  poisoned 
wound."  These  local  changes  are  soon  followed  by  marked 
constitutional  disturbance,  and  by  a  local  or  widespread  eruption 
on  the  surface  of  the  body,  at  first  papular  and  afterwards 
pustular,  and  later  there  may  form  in  the  subcutaneous  tissue 
and  muscles  larger  masses  which  soften  and  suppurate,  the  pus 
being  often  mixed  with  blood  ;  suppuration  may  occur  also  in  the 
joints.  In  some  cases  the  nasal  mucous  membrane  may  be 
secondarily  infected,  and  thence  inflammatory  swelling  may 
spread  to  the  tissues  of  the  face.  The  patient  usually  dies  in 
two  or  three  weeks,  sometimes  sooner,  with  the  symptoms  of 
rapid  pyaemia.  In  addition  to  the  lesions  mentioned,  there  may 
be  foci,  usually  suppurative,  in  the  lungs  (attended  often  with 
pneumonic  consolidation),  in  the  spleen,  liver,  bone-marrow, 


308  GLANDERS 

salivary  glands,  etc.  In  the  chronic  form  a  local  granulomatous 
condition  may  occur,  which  usually  breaks  clown  and  gives  rise  to 
the  formation  of  an  irregular  ulcer  with  thickened  margins,  and 
sanious,  often  foul,  discharge.  The  ulceration  spreads  deeply  as 
well  as  superficially,  and  the  thickened  lymphatics  also  have  a 
great  tendency  to  ulcerate,  though  the  lymphatic  system  is  not 
so  prominently  affected  as  in  the  horse.  Deposits  may  form  in 
the  subcutaneous  tissue  and  muscles,  and  the  mucous  membrane 
may  become  affected.  The  disease  may  run  a  very  chronic 
course,  lasting  for  months  or  even  years,  and  recovery  may 

occur ;  on  the  other 
hand,  such  a  case  may 
at  any  time  take  on  the 
characters  of  the  acute 
form  of  the  disease  and 
rapidly  become  fatal. 

/     /  The   Glanders   Bacil- 

lus. —  Microscopical 
Characters.  —  The  glan- 
ders bacilli  are  minute 
rods,  straight  or  slightly 
curved,  with  rounded 
ends,  and  about  the  same 
length  as  tubercle  bacilli, 
but  distinctly  thicker 

(Fig.   89).      They  show, 

FIG.  89.-Glanders  bacilli, -several  con-  however,  _  considerable 
tained  within  leucoytes,— from  peritoneal  variations  in  size  and  in 
exudate  in  a  guinea-pig  appearance,  and  their 

Stained  with  weak  carbol-fuehsm.      x  1000.  ,  ., 

protoplasm    is    otten 

broken  up  into  a  num- 
ber of  deeply-stained  portions  with  unstained  intervals  between. 
These  characters  are  seen  both  in  the  tissues  and  in  cultures, 
but,  as  in  the  case  of  many  organisms,  irregularities  in  form  and 
size  are  more  pronounced  in  cultures  (Fig.  90) ;  short  filamentous 
forms  8  to  12  /x  in  length  .are  sometimes  met  with,  but  these  are 
on  the  whole  rare.  The  organism  is  non-motile  and  does  not 
form  spores,  though  some  have  considered  certain  of  the  non- 
staining  portions  of  the  protoplasm  to  be  of  that  nature. 

Tn  the  tissues  the  bacilli  usually  occur  irregularly  scattered 
amongst  the  cellular  elements ;  a  few  may  be  contained  within 
leucocytes  and  connective-tissue  corpuscles,  but  the  position  of 
most  is  extracellular.  They  are  most  abundant  in  the  acute 
lesions,  in  which  they  may  be  found  in  considerable  numbers ; 


CULTIVATION  OF  GLANDERS  BACILLUS      309 


but  in  the  chronic  nodules,  especially  when  softening  has  taken 
place,  they  are  few  in  number,  and  it  may  be  impossible  to  find 
any  in  sections. 

Staining.  —  The  glanders  bacillus  differs  widely  from  the 
tubercle  bacillus  in  its  staining  reactions.  It  stains  with  simple 
watery  solutions  of  the  basic  stains,  but  somewhat  faintly  (better 
when  an  alkali  or  a  mor- 
dant, such  as  carbolic 
acid,  is  added),  and  even 
when  deeply  stained  it 
readily  loses  the  colour 
when  a  decolorising  agent 
such  as  alcohol  is  applied. 
We  have  obtained  the 
best  results  by  carbol- 
thionin-blue  (p.  105),  and 
we  prefer  to  dehydrate 
by  the  aniline-oil  method. 
In  film  preparations  of 
fresh  glanders  nodules 
the  bacilli  can  be  readily 
found  by  staining  with 
any  of  the  ordinary  com- 
binations, e.g.,  carbol- 
thionin-blue  or  weak  car- 
bol-fuchsin.  By  using  a 
stain  of  suitable  strength 

no  decolorising  agent  is  necessary,  the  film  being  simply  washed 
in  water,  dried,  and  mounted. 

McFadyean  recommends  that  after  sections  have  been  stained  in 
Lbffler's  methylene-blue  and  slightly  decolorised  in  weak  acetic  acid,  they 
should  be  treated  for  fifteen  minutes  with  a  saturated  solution  of  tannic 
acid  ;  thereafter  they  are  washed  thoroughly  in  water,  and  as  a  contrast 
stain  a  1  per  cent,  solution  of  acid  fuchsin  may  be  applied  for  half  a 
minute  ;  they  are  then  dehydrated,  cleared,  and  mounted.  Gram's 
method  is  quite  inapplicable,  the  glanders  bacilli  rapidly  losing  the  stain 
in  the  process. 

Cultivation. — (For  the  methods  of  separation,  vide  infra.) 
The  glanders  bacillus  grows  readily  on  most  of  the  ordinary 
media,  but  a  somewhat  high  temperature  is  necessary,  growth 
taking  place  most  rapidly  at  35°  to  37°  C.  Though  a  certain 
amount  of  growth  occurs  down  to  21°  C.,  a  temperature  above 
25°  C.  is  always  desirable. 

On  ayar  and  ylycerin-ayar  in  stroke  cultures  growth  appears 


FIG.  90.— Glanders  bacilli,  from  a  pure 
culture  on  glycerin  agar.  Stained  with 
car bol -fuchsin  and  partially  decolorised 
to  show  segmentation  of  protoplasm. 
xlOOO. 


310  GLANDERS 

along  the  line  as  a  uniform  streak  of  greyish-white  colour  arid 
somewhat  transparent  appearance,  with  moist-looking  surface, 
and  when  touched  with  a  needle  is  found  to  be  of  rather  slimy 
consistence.  Later  it  spreads  laterally  for  some  distance,  and 
the  layer  becomes  of  slightly  brownish  tint. 

On  serum  the  growth  is  somewhat  similar  but  more  transparent, 
the  separate  colonies  being  in  the  form  of  round  and  almost  clear 
drops.  In  sub-cultures  on  these  media  at  the  body  temperature 
growth  is  visible  within  twenty -four  hours,  but  when  fresh  cultures 
are  made  from  the  tissues  it  may  not  be  visible  till  the  second  day. 
Serum  or  potato,  however,  is  much  more  suitable  for  cultivating 
from  the  tissues  than  the  agar  media ;  on  the  latter  it  is  some- 
times difficult  to  obtain  growth. 

In  broth,  growth  forms  at  first  a  uniform  turbidity,  but  soon 
settles  to  the  bottom,  and  after  a  few  days  forms  a  pretty  thick 
flocculent  deposit  of  slimy  and  somewhat  tenacious  consistence. 

On  potato  at  30°  to  37°  C.  the  glanders  bacillus  nourishes 
well  and  produces  a  characteristic  appearance,  incubation  at  a 
high  temperature,  however,  being  necessary.  Growth  proceeds 
rapidly,  and  on  the  third  day  has  usually  formed  a  transparent 
layer  of  slightly  yellowish  tint,  like  clear  honey  in  appearance. 
On  subsequent  days,  the  growth  still  extends  and  becomes  darker 
in  colour  and  more  opaque,  till  about  the  eighth  day  it  has  a 
reddish-brown  or  chocolate  tint,  while  the  potato  at  the  margin 
of  the  growth  often  shows  a  greenish-yellow  staining.  The 
characters  of  the  growth  on  potato,  along  with  the  microscopical 
appearances,  are  quite  sufficient  to  distinguish  the  glanders 
bacillus  from  every  other  known  organism  (sometimes  the 
cholera  organism  and  the  b.  pyocyaneus  produce  a  somewhat 
similar  appearance,  but  they  can  be  readily  distinguished  by 
their  other  characters).  Potato  is  also  a  suitable  medium  for 
starting  cultures  from  the  tissues;  in  this  case  minute  trans- 
parent colonies  become  visible  on  the  third  day,  and  afterwards 
present  the  appearances  just  described. 

Powers  of  Resistance. — The  glanders  bacillus  is  not  killed  at 
once  by  drying,  but  usually  loses  its  vitality  after  fourteen  days 
in  the  dry  condition,  though  sometimes  it  lives  longer.  It  is 
not  quickly  destroyed  by  putrefaction,  having  been  found  to  be 
still  active  after  remaining  two  or  three  weeks  in  putrefying 
fluids.  In  cultures  the  bacilli  retain  their  vitality  for  three  or 
four  months,  if,  after  growth  has  taken  place,  they  are  kept  at 
the  temperature  of  the  room ;  on  the  other  hand,  they  are  often 
found  to  be  dead  at  the  end  of  two  weeks  when  kept  constantly 
a.t  the  body  temperature.  They  have  comparatively  feeble 


EXPERIMENTAL  INOCULATION  311 

resistance  to  heat  and  antiseptics.  Loffler  found  that  they  were 
killed  in  ten  minutes  in  a  fluid  kept  at  55°  C.,  and  in  from  two 
to  three  minutes  by  a  5  per  cent,  solution  of  carbolic  acid. 
Boiling  water  and  the  ordinarily  used  antiseptics  are  very  rapid 
and  efficient  disinfectants. 

We  may  summarise  the  characters  of  the  glanders  bacillus  by 
saying  that  in  its  morphological  characters  it  resembles  some- 
what the  tubercle  bacillus,  but  is  thicker,  and  differs  widely 
from  it  in  its  staining  reactions.  For  its  cultivation  the  higher 
temperatures  are  necessary,  and  the  growth  on  potato  presents 
most  characteristic  features. 

Experimental  Inoculation. — In  horses,  subcutaneous  injection 
of  the  glanders  bacillus  in  pure  culture  reproduces  all  the 
important  features  of  the  disease.  This  fact  was  established  at 
a  comparatively  early  date  by  Loffler  and  Schutz,  who,  after  one 
doubtful  experiment,  successfully  inoculated  two  horses  in  this 
way,  the  cultures  used  having  been  grown  for  several  generations, 
outside  the  body.  In  a  few  days  swellings  formed  at  the  sites 
of  inoculation,  and  later  broke  down  into  unhealthy-looking 
ulcers.  One  of  the  animals  died  ;  after  a  few  weeks,  the  other, 
showing  symptoms  of  cachexia,  was  killed.  In  both  animals,  in 
addition  to  ulcerations  on  the  surface  with  involvement  of  the 
lymphatics,  there  were  found,  post-mortem,  nodules  in  the  lungs, 
softened  deposits  in  the  muscles,  and  also  affection  of  the  nasal 
mucous  membrane, — nodules,  and  irregular  ulcerations.  The 
ass  is  even  more  susceptible  than  the  horse,  the  disease  in  the 
former  running  a  more  rapid  course,  but  with  similar  lesions. 
The  ass  can  be  readily  infected  by  simple  scarification  and 
inoculation  with  glanders  secretion,  etc.  (Nocard). 

Of  small  animals,  field-mice  and  guinea-pigs  are  the  most 
susceptible;  on  the  other  hand,  house-mice  and  white  mice 
enjoy  an  almost  complete  immunity.  In  field-mice,  subcutaneous 
inoculation  is  followed  by  a  very  rapid  disease,  usually  leading 
to  death  within  eight  days,  the  organisms  becoming  generalised 
and  producing  numerous  minute  nodules,  especially  in  the  spleen, 
lungs,  and  liver.  In  the  guinea-pig  the  disease  is  less  acute. 
At  the  site  of  inoculation  an  inflammatory  swelling  forms,  which 
soon  softens  and  breaks  down,  leading  to  the  formation  of  an 
irregular  crateriform  ulcer  with  indurated  margins.  The  lym- 
phatic vessels  become  infiltrated,  and  the  corresponding  lymphatic 
glands  become  enlarged  to  the  size  of  peas  or  small  nuts,  softened, 
and  semi-purulent.  The  animal  sometimes  dies  in  two  or  three 
weeks,  sometimes  not  for  a  longer  period.  Secondary  nodules, 
in  varying  numbers  in  different  cases,  may  be  present  in  the 


312  GLANDERS 

spleen,  lungs,  bones,  nasal  mucous  membrane,  testicles,  ovaries, 
etc. ;  in  some  cases  a  few  nodules  are  found  in  the  spleen  alone. 
Intraperitoneal  injection  in  the  male  guinea-pig  is  followed,  as 
pointed  out  by  Straus,  by  a  very  rapid  and  semi-purulent 
affection  of  the  tunica  vaginalis,  shown  during  life  by  great 
swelling  and  redness  of  the  testicles,  which  changes  may  be 
noticeable  in  two  or  three  days,  or  earlier  if  material  from  man 
has  been  used.  This  method  of  inoculation  has  been  found  of 
service  for  purposes  of  cultivation  and  diagnosis.  Rabbits  are 
less  susceptible  than  guinea-pigs,  and  the  effect  of  subcutaneous 
inoculation  is  somewhat  uncertain.  Accidental  inoculation  of  the 
human  subject  with  pure  cultures  of  the  bacillus  has  in  more  than 
one  instance  been  followed  by  the  acute  form  of  the  disease  and 
a  fatal  result.  •' 

Mayer  has  found  that  when  the  glanders  bacillus  is  injected  along 
with  melted  butter  into  the  peritoneum  of  a  guinea-pig,  it  shows 
filamentous,  branching,  and  club-shaped  forms ;  in  other  words,  it 
presents  the  characters  of  a  streptothrix.  Lubarsch,  on  the  other  hand, 
in  a  comparative  study  of  the  results  of  inoculation  with  acid-fast  and 
other  bacilli,  found  none  of  the  above  characters  in  the  case  of  the 
glanders  bacillus  (cf.  Tubercle,  p.  263). 

Action  on  the  Tissues. — From  the  above  facts  it  will  be  seen 
that  in  many  respects  glanders  presents  an  analogy  to  tubercle 
as  regards  the  general  characters  of  the  lesions  and  the  mode 
of  spread.  When  the  tissue  changes  in  the  two  diseases  are 
compared,  certain  differences  are  found.  The  glanders  bacillus 
causes  a  more  rapid  and  more  marked  inflammatory  reaction. 
There  is  more  leucocytic  infiltration  and  less  proliferative  change 
which  might  lead  to  the  formation  of  epithelioid  cells.  Thus 
the  centre  of  an  early  glanders  nodule  shows  a  dense  aggregation 
of  leucocytes,  most  of  which  are  poly morpho-nucl ear,  whilst  in 
the  central  parts  many  show  fragmentation  of  nuclei  with  the 
formation  of  a  deeply  staining  granular  detritus.  And  further, 
the  inflammatory  change  may  be  followed  by  suppurative 
softening  of  the  tissue,  especially  in  certain  situations,  such  as 
the  subcutaneous  tissue  and  lymphatic  glands.  The  nodules, 
therefore,  in  glanders,  as  Baumgarten  puts  it,  occupy  an 
intermediate  position  between  miliary  abscesses  and  tubercles. 
The  diffuse  coagulative  necrosis  and  caseation  which  are  so 
common  in  tubercle  do  not  occur  to  the  same  degree  in  glanders, 
and  typical  giant  cells  are  not  formed.  The  nodules  in  the  lungs 
show  leucocytic  infiltration  and  thickening  of  the  alveolar  walls, 
whilst  the  vesicles  are  filled  with  catarrhal  cells ;  there  may  also 
be  fibrinous  exudation,  whilst  at  the  periphery  of  the  nodules  con- 


MODE  OF  SPREAD  313 

nective-tissue  growth  is  present  in  proportion  to  their  age.  The 
tendency  to  spread  by  the  lymphatics  is  always  a  well-marked 
feature,  and  when  the  bacilli  gain  entrance  to  the  blood  stream, 
they  soon  settle  in  the  various  tissues  and  organs.  Accordingly, 
even  in  acute  cases  it  is  usually  quite  impossible  to  detect  the 
bacilli  in  the  circulating  blood,  though  sometimes  they  have  been 
found.  It  is  an  interesting  fact,  shown  by  observations  of  the 
disease  both  in  the  human  subject  and  in  the  horse,  as  well  as 
by  experiments  on  guinea-pigs,  that  the  mucous  membrane  of  the 
nose  may  become  infected  by  means  of  the  blood  stream — another 
example  of  the  tendency  of  organisms  to  settle  in  special  sites. 

Mode  of  Spread. — Glanders  usually  spreads  from  a  diseased 
animal  by  direct  contagion  with  the  discharge  from  the  nose  or 
from  the  sores,  etc.  So  far  as  infection  of  the  human  subject 
goes,  no  other  mode  is  known.  There  is  no  evidence  that  the 
disease  is  produced  in  man  by  inhalation  of  the  bacilli  in  the 
dried  condition.  Some  authorities  consider  that  pulmonary 
glanders  may  be  produced  in  this  way  in  the  horse,  whilst  others 
maintain  that  in  all  cases  there  is  first  a  lesion  of  the  nasal 
mucous  membrane  or  of  the  skin  surface,  and  that  the  lung  is 
affected  secondarily.  Babes,  however,  found  that  the  disease 
could  be  readily  produced  in  susceptible  animals  by  exposing 
them  to  an  atmosphere  in  which  cultures  of  the  bacillus  had 
been  pulverised.  He  also  found  that  inunction  of  the  skin 
with  vaseline  containing  the  bacilli  might  produce  the  disease, 
the  bacilli  in  this  case  entering  along  the  hair  follicles. 

Serum  Reactions. — Shortly  after  the  discovery  of  agglutination  in 
typhoid  fever,  McFadyean  showed  that  the  serum  of  glandered  horses 
possessed  the  power  of  agglutinating  glanders  bacilli.  His  later  observa- 
timis  show  that  in  the  great  majority  of  cases  of  glanders  a  1  :  50 
dilution  of  the  serum  produces  marked  agglutination  in  a  few  minutes, 
whilst  in  the  great  majority  of  non -glandered  animals  no  effect  is 
produced  under  these  conditions.  The  test  performed  in  the  ordinary 
\\.iy  is,  however,  not  absolutely  reliable,  as  exceptions  occasionally  occur 
in  both  directions,  i.e.  negative  results  by  glandered  animals  and  positive 
results  by  non-glandered  animals.  He  found  that  a  more  delicate  and 
reliable  method  is  to  grow  the  bacillus  in  bouillon  containing  a  small 
proportion  of  the  serum  to  be  tested.  In  this  way  he  obtained  a  distinct 
sediuienting  reaction  with  a  serum  which  did  not  agglutinate  at  all 
distinctly  in  the  ordinary  method.  Within  recent  times  the  sedimenta- 
tion test  by  the  ordinary  method  (p.  120)  has  been  most  generally  used. 
The  general  result  seems  to  be  that  distinct  sedimentation  within  thirty- 
six  hours  with  a  serum  dilution  of  1  : 1000  may  be  taken  as  a  positive 
result,  indicating  the  presence  of  glanders  ;  whilst  reactions  with  dilutions 
ln'twcen  this  and  1  :  500  are  highly  suspicious  but  not  conclusive.  The 
deviation  of  complement  test  (p.  130)  is  also  applicable  in  the  case  of 
glanders,  and  this  has  given  valuable  results  in  the  hands  of  various 
observers  ;  a  precipitin  reaction  mny  also  be  obtained  on  the  addition  of 


314  GLANDERS 

mallein  to  the  serum  of  a  glandered  animal.  These  reactions,  which  of 
course  depend  on  the  presence  of  anti-substance  in  the  blood  in  glanders, 
form  important  auxiliaries  to  the  method  of  diagnosis  by  means  of 
mallein. 

Mallein  and  its  Preparation.— Mallein  is  obtained  from  cultures  of  the 
glanders  bacillus  grown  for  a  suitable  length  of  time,  and,  like  tuber- 
culin, is  really  a  mixture  comprising  (1)  substances  in  the  bodies  of  the 
bacilli,  and  (2)  their  soluble  products,  not  destroyed  by  heat,  along  witli 
substances  derived  from  the  medium  of  growth.  It  was  at  first  obtained 
from  cultures  on  solid  media  by  extracting  with  glycerin  or  water,  but  is 
now  usually  prepared  from  cultures  in  glycerin  bouillon.  Such  a  culture, 
after  being  allowed  to  grow  for  three  or  four  weeks,  is  sterilised  by  heat 
either  in  the  autoclave  at  115°  C.  or  by  steaming  at  100°  C.  on  successive 
days.  It  is  then  filtered  through  a  Chamberland  filter.  The  filtrate 
constitutes  fluid  mallein.  Usually  a  little  carbolic  acid  ('5  per  cent.)  is 
added  to  prevent  it  from  decomposing.  Of  such  mallein  1  c.c.  is  usually 
the  dose  for  a  horse  (McFadyean).  Foth  has  prepared  a  dry  form  of 
mallein  by  throwing  the  filtrate  of  a  broth  culture,  evaporated  to  one- 
tenth  of  its  bulk,  into  twenty-five  or  thirty  times  its  volume  of  alcohol. 
A  white  precipitate  is  formed,  which  is  dried  over  calcium  chloride  and 
then  under  an  air-pump.  A  dose  of  this  dry  mallein  is  '05  to  '07  grm. 

The  Use  of  Mallein  as  a  Means  of  Diagnosis. — In  using  mallein  for  the 
diagnosis  of  glanders,  the  temperature  of  the  animal  ought  to  be  observed 
for  some  hours  beforehand,  and,  after  subcutaneous  injection  of  a  suitable 
dose,  it  is  taken  at  definite  intervals, — according  to  McFadyean  at  the 
sixth,  tenth,  fourteenth,  and  eighteenth  hours  afterwards,  and  on  the 
next  day.  Here  both  the  local  reaction  and  the  temperature  are  of 
importance.  In  a  glandered  animal,  at  the  site  of  inoculation  there  is  a 
somewhat  painful  local  swelling,  which  reaches  a  diameter  of  five  inches 
at  least,  the  maximum  size  not  being  attained  until  twenty-four  hours 
afterwards.  The  temperature  rises  1'5°  to  2°  C.,  or  more,  the  maximum 
generally  occurring  in  from  eight  to  sixteen  hours.  If  the  temperature 
never  rises  as  much  as  1*5°,  the  reaction  is  considered  doubtful.  In  the 
negative  reaction  given  by  an  animal  free  from  glanders,  the  rise  of 
temperature  does  not  usually  exceed  1°,  the  local  swelling  reaches  the 
diameter  of*three  inches  at  most,  and  has  much  diminished  at  the  end 
of  twenty-four  hours.  In  the  case  of  dry  mallein,  local  reaction  is  less 
marked.  Veterinary  authorities  arc  practically  unanimous  as  to  the 
great  value  of  the  mallein  test  as  a  means  of  diagnosis.  It  has  recently 
been  shown  that  mallein  instilled  into  the  conjunctival  sac,  or  inoculated 
by  scarification  into  the  skin  of  glandered  animals,  gives  corresponding 
reactions  to  the  ophthalmic  and  cutaneous  tuberculin  reactions  in  cases 
of  tuberculosis  (pp.  285,  286) ;  in  the  case  of  glanders  the  conjunctival 
reaction  would  appear  to  be  the  more  convenient  and  reliable. 

Methods  of  Examination. — Microscopic  examination  in  a 
case  of  suspected  glanders  will  at  most  reveal  the  presence  of 
bacilli  corresponding  in  their  characters  to  the  glanders  bacillus. 
An  absolute  diagnosis  cannot  be  made  by  this  method.  Cultures 
may  be  obtained  by  making  successive  strokes  on  blood  serum  or 
on  glycerin  agar  (preferably  the  former),  and  incubating  at  37°  C. 
The  colonies  of  the  glanders  bacillus  do  not  appear  till  two  days 
after.  This  method  often  fails  unless  a  considerable  number  of 


RHINOSCLEROM  A  3 1 5 

the  glanders  bacilli  are  present.  Another  method  is  to  dilute  the 
-••(Tction  or  pus  with  sterile  water,  to  varying  degrees,  and  then 
to  smear  the  surface  of  potato  with  the  mixture,  the  potatoes 
being  incubated  at  the  above  temperature.  The  colonies  on 
potatoes  may  not  appear  till  the  third  day.  The  most  certain 
method,  however,  is  by  inoculation  of  a  guinea-pig,  either  by 
subcutaneous  or  intraperitoneal  injection.  By  the  latter  method, 
as  above  described,  lesions  are  much  more  rapidly  produced,  and 
are  more  characteristic.  If,  however,  there  have  been  other 
organisms  present,  the  animal  may  die  of  a  septic  peritonitis, 
though  even  in  such  a  case  the  glanders  bacilli  will  be  found  to 
be  more  numerous  in  the  tunica  vaginalis,  and  may  be  cultivated 
from  this  situation.  It  is  extremely  doubtful  whether  the 
application  of  mallein  to  diagnosis  of  the  disease  in  the  human 
subject  is  justifiable.  There  is  a  certain  risk  that  it  may  lead 
to  the  lesions  assuming  a  more  acute  character;  moreover, 
culture  and  inoculation  tests  are  generally  available.  In  the 
case  of  horses,  etc.,  a  diagnosis  will,  however,  be  much  more 
easily  and  rapidly  effected  by  means  of  mallein,  or  by  one  of 
the  scrum  reactions  described  above.  In  some  cases  of  acute 
glanders  in  the  human  subject  the  bacillus  has  been  obtained  in 
cultures  from  the  blood  during  life. 

RHINOSCLEROMA. 

'I'll is  disease  is  considered  here  as,  from  the  anatomical 
changes,  it  also  belongs  to  the  group  of  infective  granulomata. 
It  is  characterised  by  the  occurrence  of  chronic  nodular 
thickenings  in  the  skin  or  mucous  membrane  of  the  nose,  or 
in  the  mucous  membrane  of  the  pharynx,  larynx,  or  upj)er  part 
of  the  trachea.  The  nodules  are  of  considerable  size,  sometimes 
as  large  as  a  pea ;  in  the  earlier  stages  they  are  comparatively 
smooth  on  the  surface,  but  later  they  become  shrunken  and  the 
centre  is  often  retracted.  The  disease  is  scarcely  ever  met  with 
in  this  country,  but  is  of  not  very  uncommon  occurrence  on  the 
Continent,  especially  in  Austria  and  Poland.  In  the  granulation 
tissue  of  the  nodules  there  are  to  be  found  numerous  round  and 
rather  large  cells,  which  have  peculiar  characters  and  are  often 
known  as  the  cells  of  Mikulicz.  Their  protoplasm  contains  a 
collection  of  somewhat  gelatinous  material  which  may  fill  the 
cell  and  push  the  nucleus  to  the  side.  Within  these  cells  there 
is  present  a  characteristic  bacillus,  occurring  in  little  clumps  or 
masses  chiefly  in  the  gelatinous  material.  A  few  bacilli  also  lie 
free  in  the  lymphatic  spaces  around.  This  organism  was  first 


316  GLANDERS 

observed  by  Frisch,  and  is  now  known  as  the  bacillus  of 
rhinoscleroma.  The  bacilli  have  the  form  of  short  oval  rods, 
which,  when  lying  separately,  can  be  seen  to  possess  a  distinct 
capsule,  and  which  in  all  their  microscopical  characters  correspond 
closely  with  Friedlander's  pneumobacillus.  They  are  usually  pre- 
sent in  the  lesions  in  a  state  of  purity.  It  was  at  first  stated  that 
they  could  be  stained  by  Gram's  method,  but  more  recent  obser- 
vations show  that,  like  Friedlander's  organism,  they  lose  the  stain. 

From  the  affected  tissues  this  bacillus  can  be  easily  cultivated 
by  the  ordinary  methods.  In  the  characters  of  its  growth  in 
the  various  culture  media  it  presents  a  close  similarity  to  that 
of  the  pneumobacillus,  as  it  also  does  in  its  fermentative  action 
in  milk  and  sugar-containing  fluids.  The  nail-like  appearance 
of  the  growth  on  gelatin  is  said  to  be  less  distinct,  and  the 
growth  on  potatoes  is  more  transparent  and  may  show  small 
bubbles  of  gas ;  but  it  is  doubtful  whether  any  distinct  line  of 
difference  can  be  drawn  between  the  two  organisms  so  far  as 
their  microscopical  and  cultural  characters  are  concerned. 

The  evidence  that  the  organisms  described  are  the  cause  of 
this  disease  consists  in  their  constant  presence  and  their  special 
relation  to  the  affected  tissues,  as  already  described.  From 
these  facts  alone  it  would  appear  probable  that  they  are  the 
active  agents  in  the  production  of  the  lesions.  Experimental 
inoculation  has  thrown  little  light  on  the  subject,  though  one 
observer  has  described  the  production  of  nodules  on  the  con- 
junctive of  guinea-pigs.  The  relation  of  the  rhinoscleroma 
organism  to  that  of  Friedlander  is,  however,  still  a  matter  of 
doubt,  and  the  matter  has  been  further  complicated  by  the  fact 
that  a  bacillus  possessing  closely  similar  characters  has  been 
found  to  be  very  frequently  present  in  ozcena,  and  is  often 
known  as  the  bacillus  ozcenoe.  The  last-mentioned  organism  is 
said  to  have  more  active  fermentative  powers.  From  what  has 
been  stated  it  will  be  seen  that  a  number  of  organisms,  closely 
allied  in  their  morphological  characters,  have  been  found  in  the 
nasal  cavity  in  healthy  or  diseased  conditions.  There  is  no 
doubt  that  rhinoscleroma  is  a  specific  disease  with  well-marked 
characters,  and  it  is  quite  possible  that  one  member  of  this 
group  of  organisms  may  be  the  causal  agent,  though  indis- 
tinguishable from  others  by  culture  tests.  There  is,  however, 
a  tendency  on  the  part  of  recent  investigators,  e.g.  Perkins,  to 
consider  the  "bacillus  of  rhinoscleroma"  to  be  identical  with 
the  pneumobacillus,  and  its  presence  in  the  affected  tissues  to 
represent  merely  a  secondary  invasion.  The  subject  is  one  on 
which  more  light  is  still  required, 


CHAPTER   XIII. 

ACTINOMYCOSIS  AND  ALLIED  DISEASES. 

ACTINOMYCOSIS  is  the  most  important  example  of  a  group  of 
diseases  produced  by  streptothrix  organisms.  It  is  related,  by 
the  characters  of  the  pathological  changes,  to  the  diseases  which 
have  been  described.  The  disease  affects  man  in  common  with 
certain  of  the  domestic  animals,  though  it  is  more  frequent  in 
the  latter,  especially  in  oxen,  swine,  and  horses.  The  parasite 
was  first  discovered  in  the  ox  by  Bollinger,  and  described  by 
him  in  1877,  the  name  actinomyces  or  ray  fungus  being  from  its 
appearance  applied  to  it  by  the  botanist  Harz.  In  1878  Israel 
described  the  parasite  in  the  human  subject,  and  in  the  following 
year  Ponfick  identified  it  as  being  the  same  as  that  found  in  the 
ox.  Since  that  time  a  large  number  of  cases  have  been  observed 
in  the  human  subject,  the  result  of  investigation  being  to  show 
that  it  affects  man  much  more  frequently  than  was  formerly 
supposed. 

It  is,  however,  to  be  noted  that  the  term  "actinomyces,"  as 
originally  used,  does  not  represent  one  parasite  but  a  number  of 
closely  allied  species,  as  cultures  obtained  from  various  sources 
have  presented  considerable  differences ;  and,  further,  it  is  noted 
that  other  distinct  species  of  streptothrix  have  been  cultivated 
from  isolated  cases  of  disease  in  the  human  subject  where  the 
lesions  resembled  more  or  less  closely  those  of  actinomycosis. 
In  one  or  two  instances  the  organism  has  been  found  to  be 
"  acid-fast,"  and  there  is  no  doubt  that  the  actinomyces  group 
is  closely  related  through  intermediate  forms  with  the  tubercle 
group  (vide  p.  264). 

Naked-Eye  Characters  of  the  Parasite. — The  actinomyces 
ijrows  in  the  tissues  in  the  form  of  little  round  masses  or  colonies, 
which,  when  fully  develo^d,  are  easily  visible  to  the  naked  eye, 
the  largest  being  about  the  size  of  a  small  pin's  head,  whilst  all 
si/.-s  below  this  may  be  found.  When  suppuration  is  present, 
they  lie  free  in  the  pus ;  when  there  is  no  suppuration,  they  are 

317 


318     ACTINOMYCOSIS  AND  ALLIED  DISEASES 

embedded  in  the  granulation  tissue,  but  are  usually  surrounded 
by  a  zone  of  softer  tissue.  They  may  be  transparent  or  jelly- 
like,  or  they  may  be  opaque  and  of  various  colours — white, 
yellow,  greenish,  or  almost  black.  The  appearance  depends 
upon  their  age  and  also  upon  their  structure,  the  younger  colonies 
being  more  or  less  transparent,  the  older  ones  being  generally 
opaque.  Their  colour  is  modified  by  the  presence  of  pigment 
and  by  degenerative  change,  which  is  usually  accompanied  by  a 
yellowish  coloration.  They  are  generally  of  soft,  sometimes 
tallow-like,  consistence,  though  sometimes  in  the  ox  they  are 
gritty,  owing  to  the  presence  of  calcareous  deposit.  They  may 
be  readily  found  in  the  pus  by  spreading  it  out  in  a  thin  layer 
on  a  glass  slide  and  holding  it  up  to  the  light.  They  are  some- 
times described  as  being  always  of  a  distinctly  yellow  colour, 
but  this  is  only  occasionally  the  case;  in  fact,  in  the  human 
subject  they  occur  much  more  frequently  as  small  specks  of 
semi-translucent  appearance,  and  of  greenish-grey  tint. 

Microscopical  Characters.  —  The  parasite,  which  is  now 
generally  regarded  as  belonging  to  the  streptothrix  group  of  the 
higher  bacteria  (p.  16),  presents  pleomorphous  characters.  In 
the  colonies,  as  they  grow  in  the  tissues,  three  morphological 
elements  may  be  described,  namely,  filaments,  coccus-like  bodies, 
and  clubs. 

1.  The  filaments  are  comparatively  thin,  measuring  about 
•6  /A  in  diameter,  but  they  are  often  of  great  length.  They  are 
composed  of  a  central  protoplasm  enclosed  by  a  sheath.  The 
latter,  which  is  most  easily  made  out  in  the  older  filaments  with 
granular  protoplasm,  occasionally  contains  granules  of  dark 
pigment.  In  the  centre  of  the  colony  the  filaments  interlace 
with  one  another,  and  form  an  irregular  network  which  may  be 
loose  or  dense ;  at  the  periphery  they  are  often  arranged  in  a 
somewhat  radiating  manner,  and  run  outwards  in  a  wavy  or  even 
spiral  course.  They  also  show  true  branching,  a  character 
which  at  once  distinguishes  them  from  the  ordinary  bacteria. 
Between  the  filaments  there  is  a  finely  granular  or  homogeneous 
ground  substance.  Most  of  the  colonies  at  an  early  stage  are 
chiefly  constituted  by  filaments  loosely  arranged ;  but  later,  part 
of  the  growth  may  become  so  dense  that  its  structure  cannot  be 
made  out.  This  dense  part,  starting  excentrically,  may  grow 
round  the  colony  to  form  a  hollow  sphere,  from  the  outer 
surface  of  which  filaments  radiate  for  a  short  distance  (Fig.  91). 
The  filaments  usually  stain  uniformly  in  the  younger  colonies, 
but  some,  especially  in  the  older  colonies,  may  be  segmented  so 
as  to  give  the  appearance  of  a  chain  of  bacilli  or  of  cocci,  though 


CHARACTERS  OF  THE  ACTINOMYCES 


319 


the  sheath  enclosing  them  may  generally  be  distinguished.     Rod- 
shaped  and  spherical  forms  may  also  be  seen  lying  free. 

2.  Spores  or  Gonidia. — Like  other  species  of  streptothrix,  the 
actinomyces  when  growing  on  a  culture  medium  shows  on  its 
surface  filaments  growing  upwards  in  the  air,  the  protoplasm  of 
which  becomes  segmented  into  rounded  spores  or  gonidia.  In 
natural  conditions  outside  the  body  these  gonidia  become  free, 
and  act  as  new  centres  by  growing  out  into  filaments.  They 


Flu.  91. — Actinomycosis  of  human  liver,  showing  a  colony  of  the 
parasite  composed  of  a  felted  mass  of  filaments  surrounded  by  pus. 
Paraffin  section  ;  stained  by  Gram's  method  and  with 
safranin.      x  500. 


have  somewhat  higher  powers  of  resistance  than  the  filaments, 
though  less  than  the  spores  of  most  of  the  lower  bacteria.  An 
exposure  to  75°  C.  for  half  an  hour  is  sufficient  to  kill  most 
streptothrices  or  their  spores ;  cultures  containing  spores  can 
resist  a  temperature  from  five  to  ten  degrees  higher  than  spore- 
free  cultures  (Foulerton).  It  is  probable  that  some  of  the 
spherical  bodies  formed  within  filaments  when  growing  in  the 
tissues  have  the  same  significance,  i.e.  are  gonidia,  whilst  others 
may  be  merely  the  result  of  degenerative  change.  Both  the 


320     ACTINOMYCOSIS  AND  ALLIED  DISEASES 

filaments  and  the  spherical  bodies  are  readily  stained  by  Gram's 
method. 

3.  Clubs, — These  are  elongated  pear-shaped  bodies  which  are 
seen  at  the  periphery  of  the  colony,  and  are  formed  by  a  sort 
of  hyaline  swelling  of  the  sheath  around  the  free  extremity  of 
a  filament  (Figs.  92,  93).  They  are  usually  homogeneous  and 
structureless  in  appearance.  In  the  human  subject  the  clubs  are 


FIG.  92. — Actmomyces  in  human  kidney,  showing  clnbs  radially 
arranged  and  surrounded  by  pus.  The  filaments  had  practically 
disappeared. 

Paraffin  section  ;  stained  with  hsematoxylin  and  rubin.      x  500. 

often  comparatively  fragile  structures,  which  are  easily  broken 
down,  and  may  sometimes  be  dissolved  in  water.  Sometimes 
they  are  well  seen  when  examined  in  the  fresh  condition,  but  in 
hardened  specimens  are  no  longer  distinguishable.  In  specimens 
stained  by  Gram's  method  they  are  usually  not  coloured  by  the 
violet,  but  take  readily  a  contrast  stain,  such  as  picric  acid, 
rubin,  etc.  ;  sometimes  a  darkly-stained  filament  can  be  seen 
running  for  a  distance  in  the  centre,  and  may  have  a  knob-like 
extremity.  In  many  of  the  colonies  in  the  human  subject  the 
clubs  are  absent.  In  the  ox,  on  the  other  hand,  where  there  are 


TISSUE  LESIONS  321 

much  older  colonies,  the  clubs  constitute  the  most  prominent 
feature,  whilst  in  most  colonies  the  filaments  are  more  or  less 
degenerated,  and  it  may  sometimes  be  imi>ossible  to  find  any. 
They  often  form  a  dense  fringe  around  the  colony,  and  when 
stained  by  Gram's  method  retain  the  violet  stain.  They  have, 
in  fact,  undergone  some  further  chemical  change  which  produces 
the  altered  staining  reaction.  Occasionally  in  very  chronic 


FIG.   93. — Colonies  of  actinomyces,   showing    general    structural 
arrangement  and  clubs  at  periphery.     From  pus  in  human  subject. 
Stained  Gram  and  safranin.     x  60. 


lesions  in  the  human  subject  the  clubs  stain  with  Gram's 
method.  Clubs  showing  intermediate  staining  reaction  have 
been  described  in  the  ox  by  McFadyean.  The  club-formation 
probably  represents  a  means  of  defence  on  the  part  of  the 
parasite  against  the  phagocytes  of  the  tissue ;  the  view,  formerly 
held,  that  the  clubs  are  organs  of  fructification  has  now  been 
generally  abandoned. 

Tissue  Lesions. — In  the  human  subject  the  parasite  produces 
by  its  growth  a  chronic  inflammatory  change,  usually  ending 
in  a  suppuration  which  slowly  spreads.     In  some  cases  there 
21 


322      ACTINOMYCOSIS  AND  ALLIED  DISEASES 

is  a  comparatively  large  production  of  granulation  tissue,  with 
only  a  little  softening  in  the  centre,  so  that  the  mass  feels  solid. 
This  condition  is  sometimes  found  in  the  subcutaneous  tissue, 
especially  when  the  disease  has  not  advanced  far,  and  also  in 
dense  fibrous  tissue.  In  most  cases,  however,  and  especially 
in  internal  organs,  suppuration  is  the  outstanding  feature ; 
this  is  associated  with  abundant  growth  of  the  parasite  in  the 
filamentous  form.  In  an  organ  such  as  the  liver,  multiple  foci 
of  suppuration  are  seen  at  the  spreading  margin  of  the  disease, 
often  presenting  a  honeycomb  appearance,  whilst  the  colonies 
of  the  parasite  may  be  seen  in  the  pus  with  the  naked  eye.  In 
the  older  parts  the  abscesses  have  become  confluent,  and  formed 
large  areas  of  suppuration.  The  pus  is  usually  of  greenish- 
yellow  colour,  and  of  somewhat  slimy  character. 

In  cattle  the  tissue  reaction  is  more  of  a  formative  type, 
there  being  abundant  growth  of  granulation  tissue,  which  may 
result  in  large  tumour-like  masses,  usually  of  more  or  less 
nodulated  character,  and  often  consisting  of  well-developed 
fibrous  tissue  containing  areas  of  younger  formation,  in  which, 
however,  irregular  abscess  formation  may  be  present.  The  cells 
immediately  around  the  colonies  are  usually  irregularly  rounded, 
or  may  even  be  somewhat  columnar  in  shape,  whilst  farther  out 
they  become  spindle-shaped  and  concentrically  arranged.  It  is 
not  uncommon  to  find  leucocytes  or  granulation  tissue  invading 
the  substance  of  the  colonies,  and  portions  of  the  parasite,  etc., 
may  be  contained  within  leucocytes  or  within  small  giant-cells, 
which  are  sometimes  present.  A  similar  invasion  of  old  colonies 
by  leucocytes  is  sometimes  seen  in  human  actinomycosis. 

Origin  and  Distribution  of  Lesions. — The  lesions  in  the 
human  subject  may  occur  in  almost  any  part  of  the  body,  the 
paths  of  entrance  being  very  various.  In  many  cases  the 
entrance  takes  place  in  the  region  of  the  mouth — probably 
around  a  decayed  tooth — by  the  crypts  of  the  tonsil,  or  by 
some  abrasion.  Swelling  and  suppuration  may  then  follow  in 
the  vicinity  and  may  spread  in  various  directions.  The 
periosteum  of  the  jaw  or  the  vertebrae  may  thus  become  affected, 
caries  or  necrosis  resulting,  or  the  pus  may  spread  deeply  in 
the  tissues  of  the  neck,  and  may  even  pass  into  the  mediastinum. 
Occasionally  the  parasite  may  enter  the  tissues  from  the 
oesophagus,  and  in  a  considerable  number  of  cases  the  primary 
lesion  is  in  some  part  of  the  intestine,  generally  of  the  large 
intestine.  The  parasite  penetrates  the  wall  of  the  bowel,  and 
may  be  found  deeply  between  the  coats,  surrounded  by  purulent 
material.  Thence  it  may  spread  to  the  peritoneum  or  to  the 


CULTIVATION  OF  ACTINOMYCES  323 

extraperitoneal  tissue,  the  retrocaecal  connective  tissue  and  that 
around  the  rectum  being  not  uncommonly  seats  of  suppuration 
produced  in  this  way.  A  peculiar  affection  of  the  intestine  has 
been  described,  in  which  slightly  raised  plaques  are  found  both 
in  the  large  and  small  intestines,  these  plaques  being  composed 
almost  exclusively  of  masses  of  the  actinomyces  along  with 
epithelial  cells.  This,  however,  is  a  rare  condition.  The  path 
of  entrance  may  also  be  by  the  respiratory  passages,  the  primary 
lesion  being  pulmonary  or  peribronchial ;  extensive  suppuration 
in  the  lungs  may  result.  Infection  may  also  occur  by  the  skin 
surface,  and  lastly,  by  the  female  genital  tract,  as  in  a  case 
recorded  by  Grainger  Stewart  and  Muir,  in  which  both  ovaries 
and  both  Fallopian  tubes  were  affected. 

When  the  parasite  has  invaded  the  tissues  by  any  of  these 
channels,  secondary  or  "  metastatic "  abscesses  may  occur  in 
internal  organs.  The  liver  is  the  organ  most  frequently  affected, 
though  abscesses  may  occur  in  the  lungs,  brain  (where  a  primary 
meningitis  may  also  occur),  kidneys,  etc.  In  such  cases  the 
spread  takes  place  by  the  blood  stream,  and  it  is  possible  that 
leucocytes  may  be  the  carriers  of  the  infection,  as  it  is  not 
uncommon  to  find  leucocytes  in  the  neighbourhood  of  a  colony 
containing  small  portions  of  the  filaments  in  their  interior. 

In  the  ox,  on  the  other  hand,  the  disease  usually  remains 
quite  local,  or  spreads  by  continuity.  It  may  produce  tumour- 
like  masses  in  the  region  of  the  jaw  or  neck,  or  it  may  specially 
affect  the  palate  or  tongue,  in  the  latter  producing  enlargement 
and  induration,  with  nodular  thickening  on  the  surface — the 
condition  known  as  "woody  tongue." 

Source  of  the  Parasite. — There  is  a  considerable  amount  of 
evidence  to  show  that  outside  the  body  the  parasite  grows  on 
grain,  especially  on  barley.  Both  in  the  ox  and  in  the  pig  the 
parasite  has  been  found  growing  around  fragments  of  grain 
embedded  in  the  tissues.  There  are  besides,  in  the  case  of  the 
human  subject,  a  certain  number  of  cases  in  which  there  was  a 
history  of  penetration  of  a  mucous  surface  by  a  portion  of  grain, 
and  in  a  considerable  proportion  of  cases  the  patient  has  been 
< '\ posed  to  infection  from  this  source.  The  position  of  the 
lesions  in  cattle  is  also  in  favour  of  such  a  view. 

Cultivation  (for  methods  of  isolation  see  later). — The  descrip- 
tions of  the  cultures  obtained  by  various  investigators  differ  in 
essential  particulars,  and  there  is  no  doubt  that  the  organisms 
described  are  different.  The  following  is  the  account  of  the 
organism  as  cultivated  by  Bostrb'm  : — 

On  agar  or  glycerin  ayar  at  37°  C.,    growth    is   generally 


324      ACTINOMYCOSIS  AND  ALLIED  DISEASES 


visible  on  the  third  or  fourth  day  in  the  form  of  little  trans- 
parent drops  which  gradually  enlarge  and  form  rounded  projec- 
tions of  a  reddish-yellow  tint  and  somewhat  transparent 
appearance,  like  drops  of  amber.  The  growths  tend  to  remain 
separate,  and  even  when  they  become  confluent,  the  nodular 

character  is  maintained. 
They  have  a  tough  con- 
sistence, being  with  diffi- 
culty broken  up,  and 
adhere  firmly  to  the  sur- 
face of  the  agar.  Older 
growths  often  show  on 
the  surface  a  sort  of  cor- 
rugated aspect,  and  may 
sometimes  present  the 
appearance  of  having  been 
dusted  with  a  brownish- 
yellow  powder  (Fig.  94). 

In  the  cultures  at  an 
early  stage  the  growth  is 
composed  of  branching 
filaments,  which  stain 
uniformly  (Fig.  95),  but 
later  some  of  the  super- 
ficial filaments  may  show 
segmentation  into  gonidia. 
Slight  bulbous  thicken- 
ings may  be  seen  at  the 
end  of  some  of  the  fila- 
A  B  ments,  but  true  clubs  have 

FIG.   94.— Cultures    of   the   actinomyces    on    not  been  observed, 
glycerin  agar,  of  about  three  weeks'  growth,         Qn    gelatin    the    same 
showing  the  appearances  which  occur.    The    ,       i  •      r,,i 

growth  in  A  is  at  places  somewhat  corru-    tendency  to  grow  in  little 
gated  on  the  surface.    Natural  size.  spherical  masses  is   seen, 

and  the  medium  becomes 

very  slowly  liquefied.  When  this  occurs  the  liquefied  portion 
has  a  brownish  colour  and  somewhat  syrupy  consistence,  and 
the  growths  may  be  seen  at  the  bottom,  as  little  balls,  from  the 
surface  of  which  filaments  radiate. 

The  organism  obtained  in  culture  by  Wolff  and  Israel  (vide 
infra}  is  probably  the  same  as  the  one  which  has  been  recently 
described  in  detail  by  J.  H.  Wright,  who  obtained  it  in  pure 
condition  from  fifteen  different  cases  of  the  disease.  It  differs 
markedly  from  Bostrom's  organism  in  being  almost  a  strict 


VARIETIES  OF  ACTINOMYCES 


325 


anaerobe  and  in  ceasing  to  grow  at  a  temperature  a  little  below 
that  of  the  body.  Under  ordinary  aerobic  conditions  either  no 
growth  occurs  or  it  is  of  a  very  slight  character.  On  the  surface 
of  agar  under  anaerobic  conditions  the  organism  produces  dense 
rounded  colonies  of  greyish-white  colour,  which  sometimes 
assume  a  rosette  form.  A  somewhat  curious  feature  of  growth 
is  described  by  Wright,  namely,  that  in  a  shake  culture  in 
glucose  agar  the  colonies  are  most  numerous  and  form  a  dense 
zone  about  half  an  inch  from  the  surface  of  the  medium,  that 
is,  at  a  level  where  there 
is  presumably  a  mere 
trace  of  oxygen  obtain- 
able (Fig.  96).  In 
bouillon,  growth  takes 
place  at  the  bottom  of 
the  medium  in  rounded 
masses  which  afterwards 
undergo  disintegration. 
Wright  found  when  the 
organism  was  grown  in 
the  presence  of  serum  or 
other  animal  fluids,  that 
the  formation  of  true 
clubs  occurred  at  the 
extremity  of  some  of 
the  filaments  (Fig.  97). 
From  the  conditions 
under  which  growth 
occurs,  he  is  inclined  to 
regard  it  as  a  true  para- 
site, and  doubts  whether  it  can  have  a  saprophytic  existence  out- 
side the  body,  e.g.  on  grain.  He  is  also  of  opinion  that  all  cases 
of  true  actinomycosis,  i.e.  cases  where  colonies  visible  to  the 
naked  eye  are  present,  are  probably  produced  by  one  species,  and 
that  the  aerobic  organisms  obtained  by  Bostrom  and  others  are 
probably  accidental  contaminations.  It  is  quite  evident  that 
further  investigations  are  required  in  the  light  of  the  results 
detailed.  Certainly  the  parasite  in  many  cases  of  actinomycosis 
in  the  human  subject  does  not  grow  on  ordinary  media  under 
aerobic  conditions  as  Bostrom's  organism  does. 

Varieties  of  Actinomyces  and  Allied  Forms. — It  is  probable  that  in 
the  cases  of  the  disease  described  in  the  human  subject  there  is  more  than 
one  variety  or  species  of  parasite  belonging  to  the  same  group.  Gasperini 
has  described  several  varieties  of  actinomyces  boris  according  to  the  colour 


95. — Actinomyces,    from   a  culture   on 
glycerin   agar,    snowing  the   branching   of 
the  filaments.     See  also  Plate  III.,  Fig.  10. 
Stained  with  fuchsin.      x  1000. 


326      ACTINOMYCOSIS  AND  ALLIED  DISEASES 


FIG.  96. 1 — Shake  cultures  of  actinomyces  in 
glucose  agar,  showing  the  maximum 
growth  at  some  distance  from  the  surface 
of  the  medium. 


regarded  as  a  distinct 
species.  Another  species 
was  cultivated  by  Ep- 
pinger  from  a  brain 
abscess,  and  called  by 
him  ' '  cladothrix  aste- 
roides,"  from  the  appear- 
ance of  its  colonies  on 
culture  media.  A  case 
of  general  streptothrix 
infection  in  the  human 
subject  described  by 
Mac  Donald  was  pro- 
bably due  to  the  same 
organism  as  Eppinger's. 
In  the  tissues  it  grows 
in  a  somewhat  diffuse 
manner,  and  does  not 


of  the  growths,  and  a  similar 
condition  may  obtain  in  the 
case  of  the  human  subject. 
Furthermore,  a  considerable 
number  of  strcptothrices 
have  been  found  in  cases  of 
disease  in  the  human  sub- 
ject, the  associated  lesions 
varying  in  character  from 
tubercle-like  nodules  on  the 
one  hand  to  suppurative 
processes  on  the  other.  The 
organisms  cultivated  from 
such  sources  differ  accord- 
ing to  their  microscopic 
characters  •  (for  example, 
some  form  "clubs"  whilst 
others  do  not)  according  to 
their  conditions  of  growth, 
staining  reactions,  etc.  Of 
these  only  a  few  examples 
may  here  be  mentioned,  but 
it  may  be  noted  that  the 
importance  of  the  strepto- 
th rices  as  causes  of  disease 
is  constantly  being  ex- 
tended. Wolff  and  Israel 
cultivated  from  two  cases 
of  "  actinomycosis  "  in  man 
a  streptothrix  which  differs 
in  so  many  important  points 
from  the  actinomyces  of 
Bostrom  that  it  is  now 


FIG.  97. — Section  of  a  colony  of  actinomyces 
from  a  culture  in  blood  serum,  showing  the 
formation  of  clubs  at  the  periphery,  x  1500. 


i  For    Figs.    96  and   97   we  are  indebted   to   Dr.   J.  Homer    Wright  of 
Boston,  U.S.A. 


METHODS  OF  EXAMINATION  AND  DIAGNOSIS     327 

form  clubs  ;  in  rabbits  and  guinea-pigs  it  produces  tubercle-like  lesions. 
Flexner  observed  a  streptothrix  in  the  lungs  associated  with  lesions  some- 
what like  a  rapid  phthisis,  and  applied  the  name  "pseudo-tuberculosis 
hominis  streptothricea "  ;  an  apparently  similar  condition  has  been 
described  by  Buchholz.  Berestnew  cultivated  two  species  of  streptothrix 
from  suppurative  lesions,  one  of  which  is  acid -fast  and  grows  only  in 
anaerobic  conditions.  Birt  and  Leishman  have  described  another  acid- 
fast  streptothrix  obtained  from  cirrhotic  nodules  in  the  lungs  of  a  man. 
This  organism  grows  readily  on  ordinary  media,  forming  a  white  powdery 
growth  which  afterwards  assumes  a  pinkish  colour  ;  it  is  pathogenic  for 
guinea-pigs,  in  which  it  causes  caseous  lesions.  There  is,  further,  the 
streptothrix  rnadurae  described  below. 

In  diseases  of  the  lower  animals  several  other  forms  have  been  found. 
For  example,  a  streptothrix  has  been  shown  by  Nocard  to  be  the  cause 
of  a  disease  of  the  ox, — "  farcin  du  boeuf," — a  disease  in  which  also  there 
occur  tumour-like  masses  of  granulation  tissue.  Dean  has  cultivated  from 
a  nodule  in  a  horse  another  streptothrix,  which  produces  tubercle-like 
nodules  in  the  rabbit  with  club-formation  ;  it  has  close  resemblances  to 
the  organism  of  Israel  and  Wolff.  The  so-called  diphtheria  of  calves  and 
"  bacillary  necrosis  "  in  the  ox  are  probably  both  produced  by  another 
streptotlirix  or  leptothrix,  which  grows  diffusely  in  the  tissues  in  the 
form  of  fine  felted  filaments.  Further  investigation  may  show  that  some 
of  these  or  other  species  may  occur  in  the  human  subject  in  conditions 
which  are  not  yet  differentiated. 

Experimental  Inoculation. — Inoculation  of  smaller  animals, 
such  as  rabbits  and  guinea-pigs,  has  usually  failed  to  give  positive 
results.  This  was  the  case,  for  example,  in  the  important  series 
of  experiments  by  Bostrom,  and  it  may  be  assumed  that  these 
animals  are  little  susceptible  to  the  actinomyces.  The  disease 
has,  however,  been  experimentally  produced  in  the  bovine  species 
both  by  cultures  from  the  ox  and  from  the  human  subject. 
Inoculation  with  the  organism  of  Israel  and  Wolff  produces 
nodular  lesions  both  in  rabbits  and  in  guinea-pigs,  while  Wright 
found  that  characteristic  colonies  and  lesions  resulted  although 
the  parasite  did  not  grow  to  any  great  extent.  Several  of  the 
other  species  of  streptothrix  have  been  found  to  possess  active 
pathogenic  properties. 

Methods  of  Examination  and  Diagnosis. — As  actinomycosis 
cannot  be  diagnosed  with  certainty  apart  from  the  discovery  of 
the  parasite,  a  careful  examination  of  the  pus  in  obscure  cases  of 
suppuration  should  always  be  undertaken.  As  already  stated, 
the  colonies  can  be  recognised  with  the  naked  eye,  especially 
when  some  of  the  pus  is  spread  out  on  a  piece  of  glass.  If  one 
of  these  is  washed  in  salt  solution  and  examined  unstained,  the 
clubs,  if  present,  are  at  once  seen  on  microscopic  examination.  Or 
the  colony  may  be  stained  with  a  simple  reagent  such  as  picro- 
carmine,  and  mounted  in  glycerin  or  Farrant's  solution.  To 
study  the  filaments,  a  colony  should  be  broken  down  on  a  cover- 


328      ACTIffOMYCOSIS  AND  ALLIED  DISEASES 

glass,  dried,  and  stained  with  a  simple  solution  of  any  of  the 
basic  aniline  dyes,  such  as  gentian-violet,  though  better  results 
are  obtained  by  carbol-thion in-blue,  or  by  carbol-fuchsin  diluted 
with  five  parts  of  water.  If  the  specimen  be  over-stained,  it 
may  be  decolorised  by  weak  acetic  acid.  Cover-glass  pre- 
parations of  this  kind,  and  also  of  cultures,  are  readily  stained 
by  these  methods,  but  in  the  case  of  sections  of  the  tissues, 
Gram's  method,  or  a  modification  of  it,  should  be  used  to  show 
the  filaments,  etc.,  a  watery  solution  of  acid  fuchsin  being  after- 
wards used  to  stain  the  clubs.  By  this  method,  very  striking 
preparations  may  be  obtained. 

Cultures  should  be  made  both  under  aerobic  and  anaerobic 
conditions.  Tubes  of  agar  or  glycerin  agar  should  be  inoculated 
and  incubated  at  37°  C. ;  deep  tubes  of  melted  glucose  agar 
should  also  be  used,  the  inoculated  material  being  diffused 
through  the  medium,  separate  colonies  may  thus  be  obtained. 
Owing  to  the  slow  growth  of  the  actinomyces,  however,  the 
obtaining  of  pure  cultures  is  somewhat  difficult,  unless  the  pus  is 
free  from  contamination  with  other  organisms. 

MADURA  DISEASE. 

Madura  disease  or  mycetoma  resembles  actinomycosis  both  as 
regards  the  general  characters  of  the  lesions  and  the  occurrence 
of  the  parasite  in  the  form  of  colonies  or  "granules."  There  is 
no  doubt,  however,  that  the  two  conditions  are  distinct,  and  it 
also  appears  established  that  the  two  varieties  of  Madura  disease 
(vide  infra)  are  produced  by  different  organisms.  This  disease 
is  comparatively  common  in  India  and  in  various  other  parts  of 
the  tropics  :  it  has  also  been  met  with  in  Algiers  and  in  America. 
Madura  disease  differs  from  actinomycosis  not  only  in  its  geo- 
graphical distribution  but  also  in  its  clinical  characters.  Its 
course,  for  example,  is  of  an  extremely  chronic  nature,  and 
though  the  local  disease  is  incurable  except  by  operation,  the 
parasite  never  produces  secondary  lesions  in  internal  organs. 
Vincent  also  found  that  iodide  of  potassium,  which  has  a  high 
value  as  a  therapeutic  agent  in  many  cases  of  actinomycosis,  had 
no  effect  in  the  case  of  Madura  disease  studied  by  him.  It  most 
frequently  affects  the  foot ;  hence  the  disease  is  often  spoken  of 
as  "Madura  foot."  The  hand  is  rarely  affected.  In  the  parts 
affected  there  is  a  slow  growth  of  granulation  tissue  which  has 
an  irregularly  nodular  character,  and  in  the  centre  of  the  nodules 
there  occurs  purulent  softening  which  is  often  followed  by  the 
formation  of  fistulous  openings  and  ulcers.  There  are  great 


MADURA  DISEASE 


329 


enlargement  and  distortion  of  the  part  and  frequently  caries  and 
necrosis  of  the  bones.  Within  the  softened  cavities  and  also  in 
the  spaces  between  the  fibrous  tissue,  small  rounded  bodies  or 
granules,  bearing  a  certain  resemblance  to  the  actinomyces,  are 
present.  These  may  have  a  yellowish  or  pinkish  colour,  com- 
pared from  their  appearance  to  fish  roe,  or  they  may  be  black 
like  grains  of  gunpowder,  and  may  by  their  conglomeration 
form  nodules  of  considerable  size.  Hence  a  pale  variety  and  a 
black  variety  of  the  disease  have  been  distinguished  ;  in  both 
varieties  the  granules  mentioned  reach  a  rather  larger  size  than 
in  actinomycosis.  These 
two  conditions  will  be 
considered  separately. 

Pale  Variety. — When 
the  roe-like  granules  an- 
examined  microscopically, 
tIn-\  are  found,  like  the 
actiiiomyces,  to  show  in 
their  interior  an  abundant 
mass  of  branching  fila- 
ments witli  mycelial 
arrangement.  There  may 
also  be  present  at  the 
periphery  club-like  struc- 
tures, as  in  actinomyces ; 
sometimes  they  are  ab- 
sent. These  structures 
often  have  an  elongated 
\ve<lge-shape,  forming  an 
outer  zone  to  the  colony, 

and  in  some  cases  the  filaments  can  be  found  to  be  connected  with 
them.  Vincent  obtained  cultures  of  the  parasite  from  a  case  in 
Algiers,  and  found  it  to  be  a  distinct  species :  it  is  now  known 
as  the  streptothrix  or  discomyces  Madurce.  Morphologically  it 
closely  resembles  the  actinomyces,  but  it  presents  certain  differ- 
ences in  cultural  characters.  In  gelatin  it  forms  raised  colonies 
of  a  yellowish  colour,  with  umbilication  of  the  centre,  and  there 
is  no  liquefaction  of  the  medium.  On  agar  the  growth  assumes  a 
reddish  colour ;  the  organism  flourishes  well  in  various  vegetable 
infusions  in  which"  the  actinomyces  does  not  grow.  On  all  the 
media  growth  only  takes  place  in  aerobic  conditions.  Experi- 
mental inoculation  of  various  animals  has  failed  to  reproduce  the 
There  is  therefore  no  doubt  that  the  streptothrix 
madimr  and  the  actiuomyces  are  distinct  species. 


Fie.    98.  —  Streptothrix    Madura',    showing 
branching  filaments.     From  a  culture  on 


agar. 
Stained  with  carbol-tliiomn-MiU'. 


xlOOO. 


330      ACTINOMYCOSIS  AND  ALLIED  DISEASES 

Black  Variety. —The  observations  of  J.  H.  Wright,  who 
obtained  pure  cultures  of  a  hyphomycete,  show  that  this  variety 
is  a  distinct  affection  from  the  pale  variety.  The  pigment  may 
be  dissolved  by  soaking  the  granules  for  a  few  minutes  in 
hypochlorite  of  sodium  solution,  and  the  granules  may  then  be 
crushed  out  beneath  a  cover-glass  and  examined  microscopically. 
The  granules  are  composed  of  a  somewhat  homogeneous  ground- 
substance  impregnated  with  pigment,  and  in  this  there  is  a 
mycelium  of  thick  filaments  or  hyphse,  many  of  the  segments 
of  which  are  swollen  ;  at  the  periphery  the  hyphae  form  a  zone 
with  radiate  arrangement.  In  many  of  the  older  granules  the 
parasite  is  largely  degenerated  and  presents  an  amorphous 
appearance.  Wright  planted  over  sixty  of  the  black  granules  in 
various  culture  media,  and  obtained  cultures  of  a  hyphomycete 
from  about  a  third  of  these.  The  organism  grows  well  on  agar, 
bouillon,  potato,  etc. ;  on  agar  it  forms  a  felted  mass  of  greyish 
colour,  and  in  old  cultures  black  granules  appear  amongst  the 
mycelium.  Microscopically  the  parasite  appears  as  a  mycelium 
of  thick  branching  filaments  with  delicate  transverse  septa  ;  in 
the  older  threads  the  segments  become  swollen,  so  that  strings  of 
oval-shaped  bodies  result.  No  signs  of  spore-formation  were 
noted.  Inoculation  of  animals  with  cultures  gave  negative 
results,  as  did  also  direct  inoculation  with  the  black  granules 
from  the  tissues.  Brumpt,  in  a  recent  work,  distinguishes 
several  varieties  of  parasite  concerned  in  Madura  disease,  and 
finds  that  a  pale  variety  may  be  produced  by  a  hyphomycete 
as  well  as  by  Vincent's  streptothrix ;  in  fact,  with  the  exception 
of  Vincent's  organism,  all  the  parasites  are  considered  by  him  to 
be  closely  allied  to  aspergillus. 


CHAPTER   XIV. 

ANTHRAX.1 

OTHER   NAMES. — SPLENIC     FEVER,    MALIGNANT    PUSTULE,    WOOL- 

SORTER'S  DISEASE.  GERMAN,  MILZBRAND  ;  FRENCH,  CHARBON.2 

Introductory.— Anthrax  is  a  disease  occurring  epidemically 
among  the  herbivora,  especially  sheep  and  oxen,  in  which 
animals  it  has  the  characters  of  a  rapidly  fatal  form  of 
septicaemia  with  splenic  enlargement,  attended  by  an  extensive 
multiplication  of  characteristic  bacilli  throughout  the  blood. 
The  disease  does  not  occur  as  a  natural  infection  from  man  to 
man,  but  may  be  communicated  to  him  directly  or  indirectly 
from  animals,  and  it  may  then  appear  in  one  of  three  forms. 
In  the  first  there  is  infection  through  the  skin,  in  which  a  local 
lesion,  the  "malignant  pustule,"  occurs.  In  the  second  form 
infection  takes  place  through  the  respiratory  tract.  Here  very 
aggravated  symptoms  centred  in  the  thorax,  with  rapidly  fatal 
termination,  follow.  Thirdly,  an  infection  may  occasionally 
take  place  through  the  intestinal  tract,  which  is  now  the  first 
part  to  give  rise  to  symptoms.  In  all  these  forms  of  the  affec- 
tion in  the  human  subject,  the  bacilli  are  in  their  distribution 
much  more  restricted  to  the  local  lesions  than  is  the  case  in  the 
ox,  their  growth  and  spread  being  attended  by  inflammatory 
oedema  and  often  by  haemorrhages. 

Historical  Summary. — Historical  researches  leave  little  doubt  that 
from  the  earliest  times  anthrax  has  occurred  among  cattle.  For  along 
time  its  pathology  was  not  understood,  and  it  went  by  many  names. 
Pollender  in  1849  pointed  out  that  the  blood  of  anthrax  animals  con- 
tained numerous  rod-shaped  bodies  which  he  conjectured  had  some 

1  In  even  recent  works  on  surgery  the  term  "anthrax"  maybe  found 
applied  to  any  form  of  carbuncle.  Before  its  true  pathology  was  known,  the 
local  variety  of  the  disease  which  occurs  in  man,  and  which  is  now  called 
"  malignant  pustule,"  was  known  as  "  malignant  carbuncle." 

-This  must  he  distinguished  from  "  charbou  symptomatiqiuy'  \vhu-his 
quite  a  different  disease. 

331 


332  ANTHRAX 

causal  connection  with  the  disease.  In  1863  Davaine  announced  that 
they  were  bacteria,  and  originated  the  name  bacillus  anthracis.  He  stated 
that  unless  blood  used  in  inoculation  experiments  on  animals  contained 
them,  death  did  not  ensue.  Though  this  conclusion  was  disputed,  still 
by  the  work  of  Davaine  and  others  the  causal  relationship  of  the  bacilli 
to  the  disease  had  been  nearly  established  when  the  work  of  Koch 
appeared  in  1876.  This  constituted  that  observer's  first  contribution  to 
bacteriology,  and  did  much  to  clear  up  the  whole  subject.  Koch  con- 
firmed Davaine's  view  that  the  bodies  were  bacteria.  He  observed  in  the 
blood  of  anthrax  animals  the  appearance  of  division,  and  from  this 
deduced  that  multiplication  took  place  in  the  tissues.  He  observed  them 
under  the  microscope  dividing  outside  the  body,  and  noticed  spore  forma- 
tion taking  place.  He  also  isolated  the  bacilli  in  pure  culture  outside 
the  body,  and,  by  inoculating  animals  with  them,  produced  the  disease 
artificially.  In  his  earlier  experiments  he  failed  to  produce  death  by 
feeding  susceptible  animals  both  with  bacilli  and  spores,  and  as  the 
intestinal  tract  was,  in  his  view,  the  natural  path  of  infection,  he  con- 
sidered as  incomplete  the  proof  of  this  method  of  the  spontaneous  occur- 
rence of  anthrax  in  herds  of  animals.  Koch's  observations  were,  shortly 
afterwards,  confirmed  in  the  main  by  Pasteur,  though  controversy  arose 
between  them  on  certain  minor  points.  Moreover,  further  research 
showed  that  the  disease  could  be  produced  in  animals  by  feeding  them 
with  spores,  and  thus  the  way  in  which  the  disease  might  spread 
naturally  was  explained. 

Bacillus  Anthracis. — Anthrax  as  a  disease  in  man  is  of 
comparative  rarity.  Not  only,  however,  is  the  bacillus 
anthracis  easy  of  growth  and  recognition,  but  in  its  growth  it 
illustrates  many  of  the  general  morphological  characters  of  the 
whole  group  of  bacilli,  and  is  therefore  of  the  greatest  use  to  the 
student.  Further,  its  behaviour  when  inoculated  in  animals 
illustrates  many  of  the  points  raised  in  connection  with  the 
general  pathogenic  effects  of  bacteria,  immunity,  etc.  Hence 
an  enormous  amount  of  work  has  been  done  in  investigating  it 
in  all  its  aspects. 

If  a  drop  of  blood  is  taken  immediately  after  death  from  an 
auricular  vein  of  a  cow,  for  example,  which  has  died  from 
anthrax,  and  examined  microscopically,  it  will  be  found  to  con- 
tain a  great  number  of  large  non-motile  bacilli.  On  making 
a  cover-glass  preparation  from  the  same  source,  and  staining  with 
watery  methylene-blue,  the  characters  of  the  bacilli  can  be  better 
made  out.  They  are  about  1'2  //,  thick  or  a  little  thicker,  and 
6  to  8  //,  long,  though  both  shorter  and  longer  forms  also  occur. 
The  ends  are  sharply  cut  across,  or  may  be  slightly  dimpled  so 
as  to  resemble  somewhat  the  proximal  end  of  a  phalanx.  Their 
protoplasm  is  very  finely  granular,  and  very  frequently  appears 
surrounded  by  a  capsule  whose  external  margin  is  not,  however, 
so  well  defined  as  is  the  case  with,  e.g.,  the  pneumococcus.  When 
several  bacilli  lie  end  to  end  in  a  thread,  the  capsule  seems 


BACILLUS  ANTHRACIS 


333 


common  to  the  whole  thread  (Fig.  103).     They  stain  well  with  all 
the  basic  aniline  dyes  and  are  not  decolorised  by  Gram's  method. 

Methylene  Blue  Reaction. — This  was  introduced  independently  by 
McFadyean  and  by  Heim  with  a  view  to  the  easy  recognition  of  the 
bacilli  in  blood  or  other  bodily  fluids,  and  depends  on  a  disintegration  of 
the  bacillary  capsules  which  occurs  when  these  are  imperfectly  fixed. 
Imperfect  fixation  is  attained  by  drying  a  blood  film  on  a  slide  and  hold- 
ing it  three  times  for  a  second  in  a  flame,  film  upwards  (too  great  heating 
fixes  the  capsules  and  prevents  the  reaction  from  occurring).  The  pre- 
paration is  stained  for  a  few  seconds  with  an  old  solution  of  methylene 
blue,  1  per  cent,  in  water  (i.e.,  with  a  methylene  blue  possessing  poly- 
chromatic qualities,  see  p.  113).  It  is  washed  in  water  and  dried  with 
filter  paper, — preferably  a  cover  glass  is  not  applied.  In  such  a  prepara- 
tion, between  and  near  the  bacteria  there  is  a  varying  amount  of  an 
irregularly  disposed  amorphous  or  finely  granular  material  of  a  violet  or 
reddish -purple  tint.  Frequently  the  colour  reaction  in  the  preparation 
is  so  marked  as  to  be  recognisable  naked-eye.  McFadyean  states  that 
this  reaction  does  not  occur  with  putrefactive  or  other  bacteria  which 
might  be  present  under  circumstances  where  the  recognition  of  the 
anthrax  bacilli  is  the  question  under  consideration. 

Plate  Cultures. — From  a  source  such  as  that  indicated,  it  is 
easy  to  isolate  the  bacilli  by  making  gelatin  or  agar  plates.  If, 
after  twelve  hours'  in- 
cubation at  37°  C.,  the 
latter  be  examined  under 
a  low  objective,  colonies 
will  be  observed.  They 
are  to  be  recognised  by 
1  MM ut if ul  wavy  wreaths 
like  locks  of  hair,  radiat- 
ing from  the  centre  and 
apparently  terminating 
in  u  point  which,  how- 
ever, on  examination 
with  a  higher  power,  is 
ol.xTved  to  be  a  fila- 
ment which  turns  upon 
itself  (Fig.  99).  Graham 
Smith  (vide  p.  4)  attri- 
butes the  appearance  to 
the  toughness  of  the 
bacterial  envelope,  which 

prevents  the  separation  of  individuals  from  one  another  after 
division.  Tin-  whole  colony  is,  in  fact,  probably  one  long  thread. 
Such  colonies  are  very  suitable  for  making  impression  prepara- 
tions (vide  p.  138)  which  preserve  permanently  the  appearances 


FIG.  99. — Surface  colony  of  the  anthrax 
bacillus  on  an  agar  plate,  showing  the 
characteristic  appearance,  x  30. 


334 


ANTHRAX 


described.  On  examining  such  with  a  high  power,  the  wreaths 
are  seen  to  be  made  up  of  bundles  of  long  filaments  lying 
parallel  with  one  another,  each  filament  consisting  of  a  chain 


FIG.  100. — Anthrax  bacilli,  arranged  in  chains, 
from  a  twenty-four  hours'  culture  on  agar 
at  37°  C. 

Stained  with  fuchsiu.      x  1000. 


of  bacilli  lying  end  to  end,  and  similar 
to  those  observed  in  the  blood  (Fig. 
100). 

On  gelatin  plates,  after  from  twenty- 
four  to  thirty-six  hours  at  20°  C.,  the 
same  appearances  manifest  themselves, 
and  later  they  are  accompanied  by 
liquefaction  of  the  gelatin.  In  gelatin 
plates,  however,  instead  of  the  char- 
acteristically wreathed  appearance  at 
the  margin,  the  colonies  sometimes 
give  off  radiating  spikelets  irregularly 
nodulated,  which  produce  a  star-like 
form.  These  spikelets  are  composed 
of  spirally  twisted  threads. 

From  such  plates  the  bacilli  can  be  easily  isolated,  and  the 
appearances  of  pure  cultures  on  various  media  studied. 

In  bouillon,  after  twenty-four  hours'  incubation  at  37°  C., 
there  is  usually  the  appearance  of  irregularly  spiral  threads  sus- 
pended in  the  liquid.  These,  on  being  examined,  are  seen 


FIG.  101.  Stab  culture  of 
the  anthrax  bacillus  in 
peptone-gelatin  ;  seven 
days'  growth.  It  shows 
the  "spiking,"  and  also, 
at  the  surface,  com- 
mencing liqiiefaction. 
Natural  size. 


BIOLOGY  OF  THE  B.  ANTHRACIS  335 

to  be  made  up  of  bundles  of  parallel  chains  of  bacilli.  Later, 
irmxvth  is  more  abundant,  and  forms  a  flocculent  mass  at  the 
bottom  of  the  fluid. 

In  yelatin  stab  cultures,  the  characteristic  appearance  can  be 
best  observed  when  a  low  proportion,  say  7J  per  cent.,  of  gelatin 
is  present,  and  when  the  tube  is  directly  inoculated  from 
anthrax  blood.  In  about  two  days  there  radiate  out  into  the 
medium  from  the  needle  track  numberless  very  fine  spikelets 
which  enable  the  cultures  to  be  easily  recognised.  These  spike- 
lets  are  longest  at  the  upper  part  of  the  needle  track  (Fig.  101). 
Not  much  spread  takes  place  on  the  surface  of  the  gelatin,  but 
here  liquefaction  commences,  and  gradually  spreads  down  the 
stab  and  out  into  the  medium,  till  the  whole  of  the  gelatin  may 
be  liquefied.  Gelatin  slope  cultures  exhibit  a  thick  felted 
growth,  the  edges  of  which  show  the  wreathed  appearance  seen 
in  plate  cultures.  Liquefaction  here  soon  ploughs  a  trough  in 
the  surface  of  the  medium.  Sometimes  "spiking"  does  not 
take  place  in  gelatin  stab  cultures,  only  little  round  particles  of 
growth  occurring  down  the  needle  tract,  followed  by  liquefaction. 
As  has  been  shown  by  Rd.  Muir,  this  property  of  spiking  can  be 
restored  by  growing  the  bacillus  for  twenty-four  hours  on  blood 
agar  at  37°  C.  Agar  sloped  cultures  have  the  appearance  of 
similar  cultures  in  gelatin,  though,  of  course,  no  liquefaction 
takes  place. 

Blood  serum  sloped  cultures  present  the  same  appearances  as 
those  on  agar.  The  margin  of  the  surface  growth  on  any  of  the 
solid  media  shows  the  characteristic  wreathing  seen  in  plate 
colonies.  The  occurrence  of  capsulation  of  the  bacilli  in  such 
cultures  has  been  described. 

On  potatoes  there  occurs  a  thick  felted  white  mass  of  bacilli 
showing  no  special  characters.  Such  a  growth,  however,  is  use- 
ful for  -studying  sporulation. 

The  anthrax  bacillus  will  thus  grow  readily  on  any  of  the 
ordinary  media.  It  can  usually  be  sufficiently  recognised  by  its 
microscopic  appearance,  by  its  growth  on  agar  or  gelatin  plates, 
and  by  its  growth  in  gelatin  stab  cultures.  The  growth  on 
plates  is  specially  characteristic,  and  is  simulated  by  no  other 
pathogenic  organism. 

The  Biology  of  the  B.  Anthracis. — Koch  found  that  the 
bacillus  anthracis  grows  best  at  a  temperature  of  35°  0.  Growth, 
i.e.  multiplication,  does  not  take  place  below  12°  C.  nor  above 
45°  C.  In  the  spore-free  condition  the  bacilli  have  comparatively 
low  powers  of  iv<i<t;mce.  They  do  not  stand  long  exposure  to 
60°  C.,  and  if  kept  at  ordinary  temperature  in  the  dry  condition 


336 


ANTHRAX 


they  are  usually  found  to  be  dead  after  a  few  days.  The  action 
of  the  gastric  juice  is  rapidly  fatal  to  them,  and  they  are  accord- 
ingly destroyed  in  the  stomachs  of  healthy  animals.  They  are 
also  soon  killed  in  the  process  of  putrefaction.  They  can,  how- 
ever, be  cooled  below  the  freezing-point  without  dying.  The 
bacillus  can  grow  without  oxygen,  but  some  of  its  vital  functions 
are  best  carried  on  in  the  presence  of  this  gas.  Thus  in  anthrax 
cultures  the  liquefaction  of  gelatin  always  commences  at  the 
surface  and  spreads  downwards.  Growth  is  more  rapid  in  the 
presence  of  oxygen,  and  spore  formation  does  not  occur  in  its 

absence.  The  organism 
may  be  classed  as  a  facul- 
tative anaerobe. 

Sporulation.  —  Under 
certain  circumstances 
spor  ulation  occurs  in 
anthrax  bacilli.  The 
morphological  appear- 
ances are  of  the  ordinary 
kind.  A  little  highly 
refractile  speck  appears 
in  the  protoplasm  about 
the  centre  of  the  bacillus  ; 
this  gradually  increases 
in  size  until  it  forms  an 
oval  body  about  the  same 
thickness  as  the  bacillus 
iv;no.  jn  fhp  K^lla™  ^vri 
^mf  u  '  DaClUaiy  pro- 

toplasm  (Fig.  102).  The 
latter  gradually  loses  its 
staining  capacities  and 
finally  disappears.  The 

spore  thus  lies  free  as  an  oval  highly  refractile  body  which  does 
not  stain  by  ordinary  methods,  but  which  can  be  easily  stained 
by  the  special  methods  described  for  such  a  purpose  (p.  109). 
When  the  spore  is  again  about  to  assume  the  bacillary  form  the 
capsule  is  apparently  absorbed,  and  the  protoplasm  within  grows 
out,  taking  on  the  ordinary  rod-shaped  form. 

According  to  most  observers,  sporulation  never  occurs  within 
the  body  of  an  animal  suffering  from  anthrax.  Koch  attributes 
this,  probably  rightly,  to  the  absence  of  free  oxygen.  The  latter 
gas  he  found  necessary  to  the  occurrence  of  spores  in  cultures 
outside  the  body.  Many,  however,  are  inclined  to  assign  as  the 
cause  of  sporulation  the  absence  of  the  optimum  pabulum.  Be* 


FIG.  102.—  Anthrax  bacilli  containing  spores 
(the  darkly  coloured  bodies)  ;  from  a  three 
days'  culture  on  agar  at  37°  C.  See  also 
Plate  III.,  Fig.  2. 

Stained  with  carbol-fuchsin  and  methylene- 


ANTHRAX  IN  ANIMALS  337 

sides  these  conditions  there  is  another  factor  necessary  to  sporula- 
tion,  namely,  a  suitable  temperature.  The  optimum  temperature 
tor  spore  production  is  30°  C.  Koch  found  that  spore  formation 
did  not  occur  below  18°  C.  Above  42°  C.  not  only  does 
sponilation  cease,  but  Pasteur  found  that  if  bacilli  were  kept  at 
this  temperature  for  eight  days  they  did  not  regain  the  capacity 
when  again  grown  at  a  lower  temperature.  In  order  to  make 
them  again  capable  of  sporing,  it  is  necessary  to  adopt  special 
measures,  such  as  passage  through  the  bodies  of  a  series  of 
susceptible  animals. 

Anthrax  spores  have  extremely  high  powers  of  resistance. 
In  a  dry  condition  they  will  remain  viable  for  a  year  or  more. 
Koch  found  they  resisted  boiling  for  five  minutes ;  and  dry  heat 
at  140°  C.  must  be  applied  for  several  hours  to  kill  them  with 
certainty.  Unlike  the  bacilli,  they  can  resist  the  action  of  the 
gastric  juice  for  a  long  period  of  time.  They  are  often  used 
as  test  objects  by  which  the  action  of  germicides  is  judged.  For 
this  purpose  an  emulsion  is  made  by  scraping  off  a  surface 
culture  and  rubbing  it  up  in  a  little  sterile  water.  Into  this 
sterile  silk  threads  are  dipped,  which,  after  being  dried  over 
strong  sulphuric  acid  in  a  desiccator,  can  be  kept  for  long 
periods  of  time  in  an  unchanged  condition.  For  use  they  are 
placed  in  the  germicidal  solution  for  the  desired  time,  then 
washed  with  water  to  remove  the  last  traces  of  the  reagent  and 
laid  on  the  surface  of  agar  or  placed  in  bouillon,  in  order  that  if 
death  of  the  bacilli  has  not  occurred  growth  may  be  observed 
(see  Chap.  VI.). 

Anthrax  in  Animals. — Anthrax  occurs  from  time  to  time 
epidemically  in  sheep,  cattle,  and,  more  rarely,  in  horses  and 
deer.  These  epidemics  are  found  in  various  parts  of  the  world, 
although  they  are  naturally  most  far-reaching  where  legal  pre- 
cautions to  prevent  the  spread  of  infection  are  non-existent. 
All  the  countries  of  Europe  are  from  time  to  time  visited  by  the 
disease,  but  in  some  it  is  much  more  common  than  in  others. 
In  Britain  the  death-rate  is  small,  but  in  France  the  annual 
mortality  among  sheep  was  probably  10  per  cent,  of  the  total 
number  in  the  country,  and  among  cattle  5  per  cent.  These 
figures,  however,  have  been  largely  modified  by  the  system  of 
preventive  treatment  which  will  be  presently  described.  In  sheep 
ant  I  cattle  the  disease  is  specially  virulent.  An  animal  may 
suddenly  drop  down,  with  symptoms  of  collapse,  quickening  of 
pulse  ami  ie>piiation,  and  dyspnoea,  and  death  may  occur  in 
a  few  minutes.  In  less  acute  cases  the  animal  is  apparently 
out  of  sorts,  and  does  not  feed ;  its  pulse  and  respiration  are 
22 


338  ANTHRAX 

quickened ;  rigors  occur,  succeeded  by  high  temperature ;  there 
is  a  sanguineous  discharge  from  the  bowels,  and  bloody  mucus 
may  be  observed  about  the  mouth  and  nose.  There  may  be 
convulsive  movements,  and  progressive  weakness,  with  cyanosis, 
is  followed  by  death  in  from  twelve  to  forty-eight  hours.  In 
the  more  prolonged  cases  widespread  oedema  and  extensive 
enlargement  of  lymphatic  glands  are  marked  features ;  and  in 
the  glands,  especially  about  the  neck,  actual  necrosis  with 
ulceration  may  occur,  constituting  the  so-called  anthrax  car- 
buncles. Such  subacute  conditions  are  especially  found  among 
horses,  which  are  by  nature  not  so  susceptible  to  the  disease  as 
cattle  and  sheep. 

On  post-mortem  examination  of  an  ox  dead  of  anthrax,  the 
most  noticeable  feature — one  which  has  given  the  name  "  splenic 
fever  "  to  the  disease — is  the  enlargement  of  the  spleen,  which 
may  be  two  or  three  times  its  natural  size.  It  is  of  dark-red 
colour,  and  on  section  the' pulp  is  very  soft  and  friable,  sometimes 
almost  diffluent.  A  cover-glass  preparation  may  be  made  from 
the  spleen  and  stained  with  watery  methylene-blue.  On  ex- 
amination it  will  be  found  to  contain  enormous  numbers  of 
bacilli  mixed  with  red  corpuscles  and  leucocytes,  chiefly 
lymphocytes  and  the  large  mononucleated  variety  (Fig.  103). 
Pieces  of  the  organ  may  be  hardened  in  absolute  alcohol,  and 
sections  cut  in  paraffin.  These  are  best  stained  by  Gram's 
method.  Microscopic  examination  of  such  shows  that  the 
structure  of  the  pulp  is  considerably  disintegrated,  whilst  the 
bacilli  swarm  throughout  the  organ,  lying  irregularly  amongst 
the  cellular  elements.  The  liver  is  enlarged  and  congested,  and 
may  be  in  a  state  of  acute  cloudy  swelling.  The  bacilli  are 
present  in  the  capillaries  throughout  the  organ,  but  are  not  so 
numerous  as  in  the  spleen.  The  kidney  is  in  a  similar  condition, 
and  here  the  bacilli  are  chiefly  found  in  the  capillaries  of  the 
glomeruli,  which  often  appear  as  if  injected  with  them.  The 
lungs  are  congested  and  may  show  catarrh,  whilst  bacilli  are 
present  in  large  numbers  throughout  the  capillaries,  and  may 
also  be  found  in  the  air  cells,  probably  as  the  result  of  rupture 
of  the  capillaries.  The  blood  throughout  the  body  is  usually 
fluid  and  of  dark  colour. 

The  lymphatic  system  generally  is  much  affected.  The 
glands,  especially  the  mediastinal,  mesenteric,  and  cervical 
glands,  are  enlarged  and  surrounded  by  oedematous  tissue,  the 
lymphatic  vessels  are  swollen,  and  both  glands  and  vessels  may 
contain  numberless  bacilli.  The  heart-muscle  may  be  in  a  state 
of  cloudy  swelling,  and  the  blood  in  its  cavities  contains  bacilli, 


ANTHRAX  IN  ANIMALS  339 

though  in  smaller  numbers  than  that  in  the  capillaries.  The 
intestines  are  enormously  congested,  the  epithelium  more  or  less 
desquamated,  and  the  lumen  filled  with  a  bloody  fluid.  From 
all  the  organs  the  bacilli  can  be  easily  isolated  by  stroke  cultures 
on  agar. 

It  is  important  to  note  the  existence  of  great  differences  in 
susceptibility  to  anthrax  in  different  species  of  animals.     Thus 


;^k»     -     '••-• 


Fi'i.  103. — Scraping  from  spleen  of  guinea-pig  dead  of  anthrax, 
showing  the  bacilli  mixed  with  leucocytes,  etc.  (Same  appearance 
as  in  the  ox.) 

"  Corrosive  film  "  stained  with  carl  >ol-thion  in  -blue,      x  1000. 


the  ox,  sheep  (except  those  of  Algeria,  which  only  succumb  to 
enormous  doses  of  the  bacilli),  guinea-pig,  and  mouse  are  all 
very  susceptible,  the  rabbit  slightly  less  so.  We  have  no  data  to 
determine  whether  the  disease  occurs  among  the  last  three  in  the 
wild  state.  Less  susceptible  than  this  group  are  the  horse,  deer, 
goat,  in  which  the  disease  occurs  from  time  to  time  in  nature. 
Anthrax  also  occurs  epidemically  in  the  pig,  often  from  the 
ingestion  of  the  organs  of  other  animals  dead  of  the  disease.  It 
is,  however,  doubtful  if  all  cases  of  disease  in  the  pig  described 


340  ANTHRAX 

on  clinical  grounds  as  anthrax  are  really  such.  A  careful 
bacteriological  examination  is  here  always  advisable,  especially 
of  any  oedematous  infiltration  about  the  throat,  or  in  the 
neighbouring  lymphatic  glands ;  often,  in  pigs  dying  of 
anthrax,  bacilli  may  not  occur  in  the  blood.  Any  hsemorrhagic 
infarction  in  the  spleen  of  a  suspected  animal  should  be  carefully 
investigated.  The  human  subject  may  be  said  to  occupy  a 


FIG.  104. — Portion   of  kidney  of  a  guinea-pig  dead   of  anthrax, 
showing  the  bacilli  in  the  capillaries,  especially  of  the  glomerulus. 
Paraffin  section  :  stained  by  Gram's  method  and  Bismarck-brown. 
x300. 

medium  position  between  the  highly  susceptible  and  the  rela- 
tively immune  animals.  The  white  rat  is  highly  immune  to  the 
disease,  while  the  brown  rat  is  susceptible.  Adult  carnivora  are 
also  very  immune,  and  the  birds  and  amphibia  are  in  the  same 
position. 

With  these  differences  in  susceptibility  there  are  also  great 
variations  in  the  pathological  effects  produced  in  the  natural  or 
artificial  disease.  This  is  especially  the  case  when  we  consider 
the  distribution  of  the  bacilli  in  the  bodies  of  the  less  susceptible 


ANTHRAX  IN  THE  HUMAN  SUBJECT         341 

animals.  Instead  of  the  widespread  occurrence  described  above, 
they  may  be  confined  to  the  point  where  they  first  gained  access 
to  the  body  and  the  lymphatic  system  in  relation  to  it,  or  may 
be  only  very  sparsely  scattered  in  organs  such  as  the  spleen 
(which  is  often  not  enlarged),  the  lungs,  or  kidneys.  Neverthe- 
less the  cellular  structure  of  the  organs  even  in  such  a  case  may 
show  changes,  a  fact  which  is  important  when  we  consider  the 
essential  pathology  of  the  disease. 

Experimental  Inoculation. — Of  the  animals  commonly  used 
in  laboratory  work,  white  mice  and  guinea-pigs  are  the  most 
susceptible  to  anthrax,  and  are  generally  used  for  test  inocula- 
tions. If  a  small  quantity  of  anthrax  bacilli  be  injected  into  the 
subcutaneous  tissue  of  a  guinea-pig,  a  fatal  result  follows,  usually 
within  two  days.  Post-mortem,  around  the  site  of  inoculation  the 
tissues,  owing  to  intense  inflammatory  oedema,  are  swollen  and 
gelatinous  in  appearance,  small  haemorrhages  are  often  present, 
and  on  microscopic  examination  numerous  bacilli  are  seen. 
The  internal  organs  show  congestion  and  cloudy  swelling,  with 
sometimes  small  haemorrhages,  and  their  capillaries  contain 
enormous  numbers  of  bacilli,  as  has  already  been  described  in 
the  case  of  the  ox  (Fig.  104) ;  the  spleen  also  shows  a  corre- 
sponding condition.  Highly  susceptible  animals  may  be  infected 
by  being  made  to  inhale  the  bacilli  or  their  spores,  and  also  by 
being  fed  with  spores,  a  general  infection  rapidly  occurring  by 
both  methods. 

Anthrax  in  the  Human  Subject. — As  we  have  noted,  man 
occupies  a  middle  position  in  the  scale  of  susceptibility  to 
anthrax.  It  is  always  communicated  to  him  from  animals,  and 
usually  is  seen  among  those  whose  trade  leads  them  to  handle 
the  carcases  or  skins  of  animals  which  have  died  of  the  disease. 
It  occurs  in  two  principal  forms,  the  main  difference  between 
which  is  due  to  the  site  of  entrance  of  the  organism  into  the 
lx>dy.  In  one,  the  path  of  entrance  is  through  cuts  or  abrasions 
in  the  skin,  or  through  the  hair  follicles.  A  local  condition 
called  a  "malignant  pustule"  develops,  which  may  lead  to  a 
general  infection.  This  variety  occurs  chiefly  among  butchers 
and  those  who  work  among  hides  (foreign  ones  especially).  In 
Britain  the  workers  of  the  latter  class  chiefly  liable  are  the  hide- 
porters  and  hide- workers  in  South-Eastern  London.  In  the 
other  variety  of  the  disease  the  site  of  infection  is  the  trachea 
and  bronchi,  aud  here  a  fatal  result  almost  always  follows.  The 
cause  is  the  inhalation  of  dust  or  threads  from  wool,  hair,  or 
IT! sties,  which  have  been  taken  from  animals  dead  of  the  disease, 
and  which  have  been  contaminated  with  blood  or  secretions  con- 


342  ANTHRAX 

taining  the  bacilli,  these  having  afterwards  formed  spores. 
This  variety  is  often  referred  to  as  "  woolsorter's  disease,"  from 
its  occurring  in  the  centres  of  the  woolstapling  trade  .(in 
England,  chiefly  in  Yorkshire),  but  it  also  is  found  in  places 
where  there  are  hair  and  brush  factories. 

(1)  Malignant  Pustule. — This  usually  occurs  on  the  exposed 
surfaces — the  face,  hands,  fore-arms,  and  back,  the  last  being  a 
common  site  among  hide-porters.  One  to  three  days  after 
inoculation  a  small  red  painful  pimple  appears,  soon  becoming  a 
vesicle,  which  may  contain  clear  or  blood-stained  fluid,  and  is 
rapidly  surrounded  by  an  area  of  intense  congestion.  Central 
necrosis  occurs  and  leads  to  the  malignant  pustule  proper,  which 
in  its  typical  form  appears  as  a  black  eschar  often  surrounded 
by  an  irregular  ring  of  vesicles,  these  in  turn  being  surrounded 
by  a  congested  area.  From  this  pustule  as  a  centre  subcutaneous 
oedema  spreads,  especially  in  the  direction  of  the  lymphatics; 
the  neighbouring  glands  are  enlarged.  There  is  fever,  with 
general  malaise.  On  microscopic  section  of  the  typical  pustule, 
the  central  eschar  is  noticed  to  be  composed  of  necrosed  tissue 
and  degenerating  blood  cells ;  the  vesicles  are  formed  by  the 
raising  of  the  stratum  corneum  from  the  rete  Malpighi.  Beneath 
them  and  in  their  neighbourhood  the  cells  of  the  latter  are 
swollen  and  oedematous,  the  papillae  being  enlarged  and  flattened 
out  and  infiltrated  with  inflammatory  exudation,  w^hich  also 
extends  beneath  the  centre  of  the  pustule.  In  the  tissue  next 
the  eschar  necrosis  is  commencing.  The  subcutaneous  tissue  is 
also  cedematous,  and  often  infiltrated  with  leucocytes.  The 
bacilli  exist  in  the  periphery  of  the  eschar  and  in  the  neigh- 
bouring lymphatics,  and,  to  a  certain  extent,  in  the  vesicles.  It 
is  very  important  to  note  that  widespread  oedema  of  a  limb, 
enlargement  of  neighbouring  glands,  and  fever  may  occur  while 
the  bacilli  are  still  confined  to  the  immediate  neighbourhood 
of  the  pustule.  Sometimes  the  pathological  process  goes  no 
further,  the  bacilli  gradually  die  out,  the  eschar  becomes  a  scab, 
the  inflammation  subsides,  and  recovery  takes  place.  In  the 
majority  of  cases,  however,  if  the  pustule  be  not  excised,  the 
oedema  spreads,  invasion  of  the  blood  stream  may  occur,  and 
the  patient  dies  with,  in  a  modified  degree,  the  pathological 
changes  detailed  with  regard  to  the  acute  disease  in  cattle.  In 
man  the  spleen  is  usually  not  much  enlarged,  and  the  organs 
generally  contain  few  bacilli.  The  actual  cause  of  death  is 
therefore  a  toxic  effect.  The  early  excision  of  an  anthrax 
pustule,  especially  when  it  is  situated  in  the  extremities,  is 
followed,  in  a  large  proportion  of  cases,  by  recovery. 


TOXINS  OF  THE  BACILLUS  ANTHRACIS       343 

(2)  Woolsorter's   Disease. — The    pathology   of   this   affection 
was  worked  out  in  this  country  especially  by  Greenfield.     The 
local  lesion  is  usually  situated  in  the  lower  part  of  the  trachea  or 
in  the  large  bronchi,  and  is  in  the  form  of  swollen  patches  in 
the  mucous  membrane,  often  with  haemorrhage  into  them, — small 
ulcers  may  also  be  seen.     The  tissues  are  cedematous,  and  the 
cellular  elements  are  separated,  but  there  is  usually  little  or  no 
necrosis.     There  is  enormous  enlargement  and  engorgement  of 
the  mediastinal  and  bronchial  glands,  and  haemorrhagic  infiltra- 
tion of  the  cellular  tissue  in  the  region.     There  are  pleural  and 
pericardial  effusions,  and  haemorrhagic  spots  occur  beneath  the 
serous  membranes.     The  lungs  show  great  congestion,  collapse 
and  oedema.     There  may  be  cutaneous  oedema  over  the  chest 
and  neck,  with  enlargement  of  glands,  and  the  patient  rapidly 
dies  with  symptoms  of  pulmonary  embarrassment,  and  with  a 
varying  degree  of  pyrexia.     It  is  to  be  noted  that  in  such  cases, 
though  numerous  bacilli  are  present  in  the  bronchial  lesions,  in 
the  lymphatic  glands,  and  affected  tissues  in  the  thorax,  com- 
paratively few  may  be  present  in  the  various  organs,  such  as  the 
kidney,  spleen,  etc.,  and  sometimes  it  may  be  impossible  to  find 
any. 

(3)  Infection  occasionally  takes  place  through  the  intestine, 
probably  by  ingestion  of  spores  as  in  the  case  of  animals ;  but 
this    condition  is   rare.     In   such   cases   there  occur   single   or 
multiple    local   haemorrhagic   lesions  in   the  intestinal   mucous 
membrane,  the  central  parts  of  the  haemorrhagic  areas  tending 
to  be  necrotic  and  yellowish,  and  there  may  be  a  corresponding 
affection  of  the  mesenteric  glands. 

A  considerable  number  of  cases  have  been  recorded  in  which 
hajmorrhagic  meningitis,  associated  with  the  presence  of  the 
anthrax  bacilli  in  large  numbers,  has  occurred  as  a  complica- 
tion of  various  primary  lesions. 

The  Toxins  of  the  Bacillus  Anthracis. — Various  theories 
were  formerly  held  as  to  the  mode  in  which  the  anthrax  bacillus 
produces  its  effects.  One  of  the  earliest  was  the  mechanical, 
according  to  which  it  was  supposed  that  the  serious  results  were 
produced  by  extensive  blocking  of  the  capillaries  in  the  various 
organs  by  the  bacilli.  According  to  another,  it  was  supposed 
that  the  bacilli  used  up  the  oxygen  of  the  blood,  thus  leading  to 
starvation  of  the  tissues.  In  modern  times  there  has  been  a 
tendency  to  attribute  the  effects  produced  to  toxic  action. 
Sidney  Martin  investigated  this  subject,  and  isolated  from 
cultures,  ]  'i-oto-albuinose,  deutero-albumose,  traces  of  pep- 
tone, and  alkaloidal  bodies.  By  these,  pathogenic  effects  were 


344  ANTHRAX 

produced  in  animals,  closely  similar  to  those  produced  by  the 
bacilli  themselves.  Martin,  to  account  for  the  symptoms  of  the 
disease,  considered  that  the  fever  was  mostly  due  to  the 
albumoses,  while  the  oedema  and  congestion  were  caused  by 
the  alkaloid  acting  as  a  local  irritant.  Hankin  and  Wesbrook 
arrived  at  the  conclusion  that  the  bacillus  anthracis  produces 
a  ferment  which,  diffusing  out  into  the  culture  fluid,  elaborates 
albumoses  from  the  proteids  present  in  it.  The  bacilli  also  pro- 
duce albumoses  directly  without  the  intervention  of  a  ferment. 
Marmier  isolated  from  cultures  in  peptone  solution  a  body  which 
gave  no  reactions  of  albuminoid  matter,  peptone,  propeptone, 
or  alkaloid,  and  which  he  considered  to  be  the  toxin.  It  was 
chiefly  retained  within  the  bacilli  when  these  were  growing  in 
the  most  favourable  conditions,  and  was  not  destroyed  by  heat- 
ing to  110°  C.  The  toxin  produced  by  the  b.  anthracis  growing 
in  a  fluid  medium  remains  intimately  associated  with  the 
bacterial  protoplasm,  as  such  cultures  when  filtered  are  relatively 
non-toxic. 

It  cannot  be  said  that  great  light  has  been  thrown  on  the 
pathology  of  the  disease  by  these  researches.  The  effects  of 
infection  by  the  b.  anthracis  are  those  shared  by  all  other 
organisms  producing  inflammation,  the  tendency  to  oedema- 
production  of  an  unwonted  degree  being  the  chief  special 
feature.  That  toxic  effects  do  occur  in  anthrax  is  probable, 
for  frequently,  while  the  bacilli  are  still  locally  confined,  there 
may  occur  pyrexia  and  oedema  spreading  widely  beyond  the 
pustule,  but  we  have  no  definite  information  as  to  how  these 
effects  are  produced.  In  this  disease  we  are  probably  dealing 
with  another  example  of  the  action  of  intracellular  toxins, 
regarding  which,  as  in  other  cases,  little  is  known. 

The  Spread  of  the  Disease  in  Nature. — We  have  seen  that 
the  b.  anthracis  rarely,  if  ever,  forms  spores  in  the  body,  and  if 
the  bacilli  could  be  confined  to  the  blood  and  tissues  of  carcases 
of  animals  dying  of  the  disease,  it  is  certain  that  anthrax  in  an 
epidemic  form  would  rarely  occur.  For  it  has  been  shown  by 
many  observers  that  in  the  course  of  the  putrefaction  of  such 
a  carcase  the  anthrax  bacilli  rapidly  die  out,  and  that  after  ten 
days  or  a  fortnight  very  few  remain.  But  it  must  be  remembered 
that  while  still  alive  an  animal  is  shedding  into  the  air  by  the 
bloody  excretions  from  the  mouth,  nose,  and  bowel,  myriads  of 
bacilli  which  may  rapidly  spore,  and  thus  arrive  at  a  very  re- 
sistant stage.  These  lie  on  the  surface  of  the  ground  and  are 
washed  off  by  surface  water.  At  certain  seasons  of  the  year  the 
temperature  is,  however,  sufficiently  high  to  permit  of  their 


SPREAD  OF  THE  DISEASE  IN  NATURE        345 

germination,  and  also  of  their  multiplication,  as  they  can  un- 
doubtedly grow  on  the  organic  matter  which  occurs  in  nature. 
They  can  again  form  spores.  It  is  in  the  condition  of  spores 
that  they  are  dangerous  to  susceptible  animals.  In  the  bacillary 
stage,  if  swallowed,  they  will  be  killed  by  the  acid  gastric  con- 
tents ;  but  as  spores  they  can  pass  uninjured  through  the 
stomach,  and  gaining  an  entrance  into  the  intestine,  infect  its 
wall,  and  ultimately  reach,  and  multiply  in  the  blood.  It  is 
known  that  in  the  great  majority  of  cases  of  the  disease  in  sheep 
ami  oxen,  infection  takes  place  thus  from  the  intestine.  It  was 
thought  by  Pasteur  that  worms  were  active  agents  in  the  natural 
spread  of  the  disease  by  bringing  to  the  surface  anthrax  spores. 
Koch  made  direct  experiments  on  this  point,  and  could  get  no 
evidence  that  such  was  the  case.  He  thinks  it  much  more 
probable  that  the  recrudescence  of  epidemics  in  fields  where 
anthrax  carcases  have  been  buried  is  due  to  persistence  of  spores 
on  the  surface  which  has  been  infected  by  the  cattle  when  alive. 
In  Britain  it  is  common  to  attribute  the  occurrence  of  sporadic 
outbreaks  to  infection  by  imported  feeding  stuffs.  Scientific 
proof  of  such  a  method  of  infection  being  common  is  still 
wanting, 

The  Disposal  of  the  Carcases  of  Animals  dead  of  Anthrax. — It  is  ex- 
tremely important  that  anthrax  carcases  should  be  disposed  of  in  such  a, 
way  as  to  prevent  their  becoming  future  sources  of  infection.  If  anthrax 
be  suspected  as  the  cause  of  death,  no  post-mortem  examination  should  be 
made,  but  only  a  small  quantity  of  blood  removed  from  an  auricular 
vein  for  bacteriological  investigation.  If  such  a  carcase  be  now  buried 
in  a  deep  pit  surrounded  by  quicklime,  little  danger  of  infection  will  be 
run.  The  bacilli  being  conftned  within  the  body  will  not  spore,  and  will 
die  during  the  process  of  putrefaction.  The  danger  of  sporulation  taking 
place  is,  of  course,  much  greater  when  an  animal  has  died  of  an  unknown 
disease,  which,  on  post-mortem  examination,  has  proved  to  be  anthrax, 
but  similar  measures  for  burial  must  be  here  adopted.  In  some  countries 
anthrax  carcases  are  burned,  and  this,  if  practicable,  is  of  course  the  best 
means  of  treating  them.  The  chief  source  of  danger  to  cattle  subsequently, 
however,  proceeds  from  the  infection  of  fields,  yards,  and  byres  with  the 
offal  and  the  discharge  from  the  mouths  of  anthrax  animals.  All  material 
that  can  be  recognised  as  such  should  be  burned  along  with  the  straw  in 
which  the  animals  have  lain.  The  stalls  or  buildings  in  which  the 
anthrax  cases  have  been  must  be  limewashed.  Needless  to  say,  the 
greatest  care  must  be  taken  in  the  case  of  men  who  handle  the  animal  or 
its  carcase  that  they  have  no  wounds  on  their  persons,  and  that  they 
thoroughly  disinfect  themselves  by  washing  their  hands,  etc.,  in  1  to 
1000  solution  of  corrosive  sublimate  or  lysol,  and  that  all  clothes  soiled 
with  blood,  etc.,  from  anthrax  animals  be  thoroughly  boiled  or  steamed 
for  half  an  hour  before  being  washed. 

The   Immunising  of  Animals   against  Anthrax. — Having 


346  ANTHRAX 

ascertained  that  there  was  ground  for  believing  that  in  cattle 
one  attack  of  anthrax  protected  against  a  second,  Pasteur  (in 
the  years  1880-82)  elaborated  a  method  by  which  a  mild  form 
of  the  disease  could  be  given  to  animals,  which  rendered 
harmless  a  subsequent  inoculation  with  virulent  bacilli.  He 
found  that  the  continued  growth  of  anthrax  bacilli  at  42°  to 
43°  C.  caused  them  to  lose  their  capacity  of  producing  spores, 
and  also  gradually  to  lose  their  virulence,  so  that  after  twenty- 
four  days  they  could  no  longer  kill  either  guinea-pigs,  rabbits, 
or  sheep.  Such  cultures  constituted  his  premier  vaccin,  and 
protected  against  the  subsequent  inoculation  with  bacilli  which 
had  been  grown  for  twelve  days  at  the  same  temperature,  and 
the  attenuation  of  which  had  therefore  not  been  carried  so  far. 
The  latter  constituted  the  deuxieme  vaccin.  It  was  further 
found  that  sheep  thus  twice  vaccinated  now  resisted  inoculation 
with  a  culture  which  usually  would  be  fatal.  The  method  was 
to  inoculate  a  sheep  on  the  inner  side  of  the  thigh  by  the 
subcutaneous  injection,  from  a  hypodermic  syringe,  of  about 
five  drops  of  the  premier  vaccin  \  twelve  days  later  to  again 
inoculate  with  the  deuxieme  vaccin ;  fourteen  days  later  an 
ordinary  virulent  culture  was  injected  without  any  ill  result. 
This  method  was  applicable  also  to  cattle  and  horses,  about 
double  the  dose  of  each  vaccine  being  here  necessary.  Extended 
experiments  in  France  generally  confirmed  earlier  results,  and 
the  method  was,  before  long,  used  to  mitigate  the  disease,  which 
in  many  departements  was  endemic  and  a  very  great  scourge. 
Since  that  time  the  method  has  been  regularly  in  use.  It  is 
difficult  to  arrive  at  a  certain  conclusion  as  to  its  merits. 
Undoubtedly  a  certain  number  of  animals  die  of  anthrax  either 
after  the  first  or  second  vaccination,  or  during  the  year  following 
vaccination.  At  the  end  of  a  year  the  immunity  is  lost  in 
about  40  per  cent,  of  the  animals  vaccinated  ;  and  thus  to  be 
permanently  efficacious  the  process  would  have  to  be  repeated 
every  year.  Further,  the  immunity  is  much  higher  in  degree 
if,  after  the  first  and  second  vaccinations,  an  inoculation  with 
virulent  anthrax  is  performed.  Everything  being  taken  into 
account,  however,  there  is  no  doubt  that  the  mortality  from 
natural  anthrax  is  much  diminished  by  this  system. 

During  the  twelve  years  1882-93,  3,296,815  sheep  were  vaccinated, 
with  a  mortality,  either  after  the  first  or  second  vaccination,  or  during 
the  subsequent  twelve  months,  of  0*94  per  cent.,  as  contrasted  with  the 
ordinary  mortality  in  all  the  flocks  of  the  districts  of  10  per  cent. 
During  the  same  time  438,824  cattle  were  vaccinated,  \vith  a  mortality, 
of  0*34  per  cent.,  as  contrasted  with  a  probable  mortality  of  5  per  cent,  if 
they  had  been  unprotected. 


IMMUNISATION  AGAINST  ANTHRAX          347 

The  immunisation  of  animals  against  anthrax  has  always 
been  found  to  be  a  difficult  proceeding.  The  most  usual 
technique  has  been  to  commence  with  Pasteur's  vaccines,  and  to 
follow  these  by  careful  dosage  with  virulent  cultures.  Marchoux 
in  this  way  produced  immunity,  and  found  that  the  serum  of 
immune  animals  had  a  certain  degree  of  protective  and  curative 
action.  The  most  successful  attempts  in  this  direction  have 
been  those  of  Sclavo  and  of  Sobernheim.  The  former  observer, 
after  trying  various  animals,  came  to  the  conclusion  that  the 
ass  was  the  most  suitable.  He  first  employed  a  method  similar 
to  that  of  Marchoux ;  later,  however,  after  noting  the  effects 
of  the  serum  of  an  animal  so  immunised,  he  commenced  the 
immunisation  by  injecting  5  to  15  c.c.  of  this  serum  along  with 
a  slightly  attentuated  culture  of  the  bacilli.  A  few  days  later 
this  was  followed  up  with  injections  of  virulent  cultures  which 
could  now  be  periodically  introduced  for  many  months,  and  a 
high  degree  of  immunity  resulted.  What  was  even  more 
important,  the  serum  of  such  an  animal  had  strongly  protective 
and  curative  properties.  It  has  been  extensively  used  in  the 
treatment  of  anthrax  in  man.  In  a  case  of  malignant  pustule 
30  to  40  c.c.  are  injected  in  quantities  of  10  c.c.  into  the 
abdominal  wall,  and  if  necessary  the  injection  is  repeated  on  the 
following  day.  In  cases  treated  by  Sclavo  himself  the  serum  is 
alone  employed,  and  its  action  is. not  aided  by  the  excision 
of  the  pustule  usually  practised.  The  results  obtained  have  been 
very  good, — Sclavo,  out  of  164  cases,  had  only  ten  deaths  or 
about  a  fourth  of  the  ordinary  mortality  in  Italy.  Sobernheim 
independently  elaborated  an  almost  identical  method  of  com- 
bining passive  with  active  immunisation  for  the  obtaining  of  a 
powerful  anti-serum,  and  he  has  used  this  for  the  protective 
inoculation  of  cattle.  The  technique  is  to  inject  a  mixed  serum 
obtained  from  the  ox,  the  horse,  and  the  sheep,  into  one  side 
of  the  neck  or  into  one  thigh  and  the  culture  (Pasteur's  second 
vaccine)  into  the  other  side ;  the  doses  given  are  for  cattle  or 
horses  5  c.c.  of  serum  and  0'5  c.c.  culture,  and  for  sheep  4  c.c. 
of  serum  and  0*25  c.c.  culture.  The  method  has  been  widely 
used  in  Germany  and  in  Brazil,  and  its  originator  claims  as  its 
advantages  simplification  of  application,  in  that  one  operation 
instead  of  two  is  sufficient,  less  risk  of  death  following  the 
immunisation  procedure,  and  higher  degree  and  more  lasting 
character  of  the  immunity  resulting.  During  the  development 
of  active  immunity  it  is  likely  in  every  case  (see  Immunity)  that 
there  is  a .  period  of  increased  susceptibility  to  the  disease. 
Such  a  period  would  be  more  likely  to  occur  with  the  Pasteur 


348  ANTHRAX 

method  than  with  the  Sobernheim  procedure,  where  the 
presence  in  the  animal's  body  of  the  protective  serum  might  tide 
it  over  the  stage  when  the  action  of  the  vaccine  was  lowering 
its  resistance. 

The  effects  of  the  b.  anthracis  have  been  much  studied  with 
a  view  to  the  shedding  of  light  on  the  processes  obtaining  in 
resistance  and  the  development  of  immunity.  Many  puzzling 
facts  have  long  been  known ;  for  example,  in  the  dog,  which 
shows  great  natural  resistance,  the  serum  has  little  if  any 
bactericidal  action,  while  in  the  susceptible  rabbit  ther.e  is 
present  a  serum  capable  of  killing  the  organism.  Such  observa- 
tions have  hitherto  been  without  explanation.  Again,  the 
properties  of  the  serum  of  immune  animals  have  been  much 
discussed.  Sobernheim  and  others  have  been  unable  to  detect 
in  it  any  trace  of  special  bactericidal  action.  Sclavo  found  that 
the  serum  when  heated  to  55°  C.  did  not  lose  its  protective 
properties;  as  the  serum  might  have  been  complemented  (see 
Immunity)  by  the  serum  of  the  animal  into  which  it  was  injected, 
he  simultaneously  introduced  an  anti-complementary  serum  and 
found  that  the  heated  serum  was  still  effectual.  From  this  he 
deduces  that  in  the  action  of  the  serum  substances  of  the  nature 
of  immune  body  and  complement  are  not  concerned.  Many 
have  thought  that  the  serum  had  a  stimulating  effect  on  the 
leucocytes,  but  Cler  has  brought  forward  ground  for  supposing 
that  its  effect  is  a  sensitising  one  on  the  bacteria,  and  that  thus 
the  effects  are  to  be  traced  to  opsonic  action.  With  regard  to 
the  formation  of  the  protective  substances,  it  is  stated  that  the 
spleen  and  bone-marrow  are  richer  in  these  than  the  blood  fluids. 
In  this  connection  an  interesting  fact  may  be  mentioned,  namely, 
that  Roger  and  Gamier  found  evidence  of  the  liver  and  spleen 
having  special  capacities  for  killing  anthrax  bacilli ;  an  otherwise 
fatal  dose  could  be  introduced  into  the  portal  vein  or  the  splenic 
artery'  without  causing  death.  It  has  been  thought  that  the 
capsule  frequently  developed  by  the  anthrax  bacillus  is  a  defen- 
sive mechanism  against  bactericidal  and  bacteriolytic  capacities 
in  an  infected  animal.  It  is  stated  that  capsulation  renders  the 
bacillus  less  susceptible  to  phagocytosis.  Opinion  on  the  signific- 
ance of  capsule  formation  is  at  present  divided. 

Methods  of  Examination. — These  include  (a)  microscopic 
examination ;  (b)  the  making  of  cultures ;  and  (c)  test  in- 
oculations. 

(a)  Microscopic  Examination. — In  a  case  of  suspected 
malignant  pustule,  film  preparations  should  be  made  from  the 
fluid  in  the  vesicles  or  from  a  scraping  of  the  incised  or  excised 


METHODS  OF  EXAMINATION  349 

pustule,  and  stained  with  a  watery  solution  of  methylene-blue 
and  also  by  Gram's  method.  By  this  method  practically  con- 
clusive evidence  may  be  obtained;  but  sometimes  the  result 
is  doubtful,  as  the  bacilli  may  be  very  few  in  number. 
McFadyean's  methylene-blue  method  (p.  333)  should  also  be 
applied.  In  all  cases  confirmatory  evidence  should  be  obtained 
by  culture.  Occasionally  bacilli  are  so  scanty  that  both  film 
preparations  made  from  different  parts  and  even  cultures  may 
give  negative  results,  and  yet  a  few  bacilli  may  be  found  when 
a  section  of  the  pustule  is  examined.  It  should  be  noted  that 
the  greatest  care  ought  to  be  taken  in  manipulating  a  pustule 
before  excision,  as  the  diffusion  of  the  bacilli  into  the 
surrounding  tissues  may  be  aided  and  the  condition  greatly 
aggravated.  The  examination  of  the  blood  in  cases  of  anthrax 
in  man  usually  gives  negative  results,  with  the  exception  of  very 
severe  cases,  when  a  few  bacilli  may  be  found  in  the  blood 
shortly  before  death,  though  even  then  they  may  be  absent. 

(h]  Cultivation. — A  small  quantity  of  the  material  used  for 
microscopic  examination  should  be  taken  on  a  platinum  needle, 
and  successive  strokes  made  on  agar  tubes,  which  are  then 
incubated  at  37°  C.  At  the  end  of  twenty-four  hours  anthrax 
colonies  will  appear,  and  can  be  readily  recognised  from  their 
wavy  margins  by  means  of  a  hand  lens.  They  should  also  be 
examined  microscopically  by  means  of  film  preparations. 

(c)  Test  Inoculation. — A  little  of  the  suspected  material 
should  be  mixed  with  some  sterile  bouillon  or  water,  and 
injected  subcutaneously  into  a  guinea-pig  or  mouse.  If  anthrax 
bacilli  are  present,  the  animal  usually  dies  within  two  days,  with 
the  changes  in  internal  organs  already  described.  The  diagnosis 
of  an  organism  as  the  anthrax  bacillus  cannot  be  said  to  be 
substantiated  till  its  pathogenicity  has  been  proved. 


CHAPTEE  XV. 

TYPHOID  FEVER— BACILLI  ALLIED  TO  THE 
TYPHOID  BACILLUS. 

Introductory. — The  organism  now  known  as  the  bacillus 
typhosus  was  first  described  in  1880-1  by  Eberth,  who  observed 
its  microscopic  appearance  in  the  intestinal  ulcers  and  in  the 
spleen  in  cases  of  typhoid  fever  (German,  Abdominaltyphus). 
It  was  first  isolated  (from  the  spleen)  in  1884  by  Gaffky,  and  its 
cultural  characters  were  then  investigated.  In  1885  Escherich 
observed  a  bacillus,  now  known  as  the  bacillus  coli  communis, 
which  occurs  in  the  normal  intestine,  and  which  both  micro- 
scopically and  culturally  closely  resembles  the  typhoid  bacillus. 
Ordinarily  the  b.  coli  is  no  doubt  a  harmless  saprophyte,  but 
under  experimental  conditions  in  animals  and  also  naturally  in 
man  it  may  manifest  pathogenic  properties.  Investigation  has 
shown  that  these  two  bacilli  belong  to  a  widespread  group 
of  organisms  isolated  from  various  disease  conditions,  chiefly 
of  the  intestine,  which  all  bear  close  resemblances  to  one 
another,  and  whose  differentiation  is  often  a  matter  of  consider- 
able difficulty.  Other  members  of  this  group  are  the  para- 
typhoid bacillus,  the  organism  of  bacillary  dysentery,  the  b. 
enteritidis  of  Gaertner,  the  psittacosis  bacillus,  and  the  bacillus 
of  hog  cholera. 

The  general  characters  of  the  group  are  as  follows :  the 
organisms,  which  are  microscopically  indistinguishable,  are  thin 
non-sporing  bacilli,  which  in  cultures  often  show  variation  in 
length ;  they  are  mostly  motile,  but  this  quality  varies  in  differ- 
ent members ;  they  possess  flagella  springing  from  all  round 
the  bacillus ;  they  stain  with  ordinary  dyes,  and  are  all  Gram- 
negative  ;  they  are  all  facultative  anaerobes,  i.e.  they  grow  best 
in  the  presence  of  oxygen,  but  can  tolerate  its  absence ;  in 
growth  characters  on  ordinary  media  they  closely  resemble  one 
another,  and,  generally  speaking,  they  do  not  liquefy  gelatin; 
they  show  wide  differences  in  fermentative  capacities,  and  are 
chiefly  to  be  distinguished  by  their  immunity  reactions. 

350 


BACILLUS  COLI  COMMUNIS 


351 


THE  BACILLUS  COLI  COMMUNIS. 

Although  the  discovery  of  the  bacillus  coli  communis  was 
subsequent  to  that  of  the  bacillus  typhosus,  it  is  convenient  to 
commence  with  a  description  of  the  former,  as  it  presents  more 
positive  characters  than  any  other  member  of  the  group  to  which 
it  belongs. 

Bacillus  Coli  Communis. — Morphological  Cliaracters. — These 
are  best  seen  in  cultures.  The  bacillus  is  ordinarily  from  2  to 
4  p.  long  and  about  *5  /A  broad ;  longer  forms  up  to  8  or  10  /A 
are  not  infrequent  (Fig.  105).  It  is  usually  found  to  be  motile, 
but  the  motility  varies 
in  different  strains  and 
under  different  growth 
conditions  in  the  same 
strain.  Here  it  is  best 
to  use  bouillon  cultures 
incubated  at  37°  C.  for 
from  six  to  twelve 
hours.  The  organism 
may  stain  somewhat 
faintly  with  watery  dyes, 
but  is  readily  demon- 
strated with  carbol- 
fuchsin  even  in  fairly 
\\vak  solution  (1  of  the 
Ziehl-Neelsen  stain  in 
20  of  water) ;  it  is 
Gram-negative.  By  ap- 
propriate staining  1).  coli 
derived  from  cultures 

can  be  shown  to  possess  fiagella  springing  from   all  round   the 
organism,  varying  in  number  and  occasionally  rather  short. 

Culture  Reactions  on  Ordinary  Media. — The  following  are 
the  appearances  of  the  b.  coli  in  the  ordinary  culture  media : — 

In  bouillon,  it  produces  a  uniform  turbidity.  When  grown  in 
fluid  gelatin,  it  is  stated  by  Klein  to  tend  to  form  flocculi  floating 
on  the  surface  rather  than  a  uniform  turbidity.  In  stab  cultures 
on  jteptoue  yelatin  an  abundant  film-like  growth  takes  place  on 
the  surface,  and  there  is  a  whitish  or  brownish-white  line  along 
the  stall.  No  liquefaction  of  the  gelatin  occurs,  but  occasionally 
a  few  bubbles  of  gas  develop  (Fig.  109,  C).  In  sloped  cigar 
tni>i>*  a  somewhat  dense,  glistening,  white  or  brownish- white 
growth  occurs  along  the  stroke.  When  ayar  jjlates  are  used 


Fie.    105. — Bacillus    coli   communis.      Film 

preparation  from  a  young  culture  on  agar. 
Stained  with  weak  carbol-tuchsin.      x  1000. 


352  TYPHOID  FEVER 

for  the  separation  of  the  organism,  the  surface  colonies  are 
somewhat  large,  and  it  may  be  irregular  in  outline,  but 
the  deep  colonies  are  smaller  and  lenticular  in  shape,  and 
under  a  low  power  of  the  miser oscope  appear  rather  dense  to 
transmitted  light.  A  similar  growth  occurs  on  blood  serum. 
On  potatoes,  in  forty-eight  hours,  there  is  a  distinct  film  of 
growth  of  a  brownish  tint,  sometimes  with  a  moist  surface, 
which  rapidly  spreads  and  becomes  thicker.  The  appearance 
on  potato,  however,  varies  much  with  the  different  strains  and 
also  with  the  reaction  of  the  potato. 

Culture  Reactions  on  Special  Media. — A  great  variety  of 
media  have  been  used  for  the  appreciation  of  special  characters 
in  the  b.  coli.  These  reactions  depend  upon  the  capacities  of  the 
organism  to  originate  chemical  changes  in  a  variety  of  substances. 

A.  Fermentative  Reactions  on  Carbo-hydrates. — B.  coli  shows 
great  powers  of  splitting  up  carbo-hydrates  with  the  formation 
of  acids,  especially  lactic  acid,  and  gas,  chiefly  carbon  dioxide 
and  hydrogen. 

Gelatin  Shake  Cultures. — If  a  tube  of  gelatin  be  melted, 
infected  with  b.  coli,  shaken  up,  allowed  to  solidify,  and  kept 
at  room  temperature,  distinct  growth  of  the  organism  occurs, 
and  round  each  little  colony,  bubbles  of  gas  form,  which  in  time 
coalesce  and  give  the  tube  a  readily  recognised  appearance. 
This  phenomenon  is  due  to  the  organism  fermenting  the  sugars 
originally  present  in  the  meat,  and  is  thus  to  be  grouped  with 
other  carbo-hydrate  reactions. 

Fermentation  of  Sugars. — As  stated  on  page  80,  litmus  or 
neutral-red  peptone  water,  or  dextrose-free  bouillon  in  Durham's 
tubes  is  used,  the  sugar  to  be  employed  being  added  in  the 
proportion  of  half  to  .  one  per  cent.  The  fermentative  capacities 
of  the  b.  coli  are  very  wide.  It  produces  acid  and  gas  in 
glucose,  lactose,  laevulose,  galactose,  maltose,  raffinose,  mannite, 
dulcite,  sorbite,  and  very  frequently  in  cane  sugar  (saccharose).1 
It  produces  a  similar  change  in  the  glucosides,  salicin,  and 
arbutin. 

The  reactions  of  b.  coli  in  some  media  other  than  simple 
sugar  solutions  likewise  depend  on  sugar  fermentation,  and  of 
these  are  the  following  : — 

Curdling  of  Milk. — If  the  b.  coli  be  grown  in  milk,  preferably 
litmus  milk,  acid  is  produced  from  the  lactose  present  which 
further  curdles  the  milk.  If  litmus  milk  be  used,  the  acid 
reaction  should  be  permanent  when  growth  is  allowed  to  go  on 

1  A  strain  of  b.  coli  fermenting  cane   sugar  was  formerly  referred  to  as 
b.  coli  comnmnior,  but  this  differentiating  term  has  been  discarded. 


CULTURE  REACTIONS  ON  SPECIAL  MEDIA     353 

for  some  days.     A  similar  reaction  is  observed  if  litmus  whey  is 
used  (p.  51). 

Measuring  of  Gas  Formation. — As  has  been  said,  the  gases  produced 
by  the  b.  coli  in  fermenting  sugars  are  chiefly  carbon  dioxide  and 
hydrogen.  Many  observers  attach  considerable  importance,  first,  to  the 
amount  of  gas  formed  from  a  given  quantity  of  glucose  in  a  given  time, 
and,  second,  to  the  ratios  of  the  two  gases  to  one  another,  in  such  a 
fermentation.  For  the  observation  of  this,  MacConkey  recommends  the 
following  method  :  fermentation  tubes  (p.  81,  Fig.  36,  c),  with  the 
closed  limb  graduated,  containing  2  per  cent,  peptone  (Witte)  and  1  per 
cent,  glucose  in  tap  water,  are  inoculated  and  incubated  for  forty-eight 
hours  at  37°  C.  The  tube  is  allowed  to  cool  and  the  total  amount  of  gas 
noted.  The  bulb  is  then  filled  with  2  per  cent,  sodium  hydrate  solution, 
the  opening  closed  with  the  thumb  and  thoroughly  shaken.  After  the 
gas  has  been  collected  in  the  closed  arm  the  thumb  is  removed  and  the 
ratio  of  the  hydrogen  left  to  the  original  gas  volume  is  read  off. 

Voges  and  Proskauer's  Reaction. — This  is  a  reaction  which  is 
not  given  by  the  classical  type  of  b.  coli,  but  as  it  occurs  with 
many  members  of  the  coli  group  it  may  be  described  here.  It 
also  depends  on  carbo-hydrate  fermentation.  A  glucose  peptone 
solution  tube  is  inoculated  and  growth  allowed  to  take  place  for 
three  days.  A  solution  of  caustic  potash  is  added  and  the  tube 
allowed  to  stand  for  twenty-four  hours  at  room  temperature.  A 
red  fluorescent  colour  is  produced,  causing  the  medium  to 
resemble  a  weak  alcoholic  solution  of  eosin. 

B.  Action  on  Neutral-Red. — When  b.  coli  is  grown  on  neutral- 
red  lactose  bouillon,  a  rosy  red  colour,  the  effect  of  the  lactic 
acid  upon  the  dye,  is  at  first  seen.     Frequently  this  is  succeeded 
by  the  appearance  of  a  green  fluorescence  due  to  a  direct  action 
of  the  organism  upon   the  dye.     This  is  evidenced  by  the  fact 
that  the  neutralisation  of  the  lactic  acid  by  an  alkali  does  not 
lead  to  a   reproduction    of   the   original   alkaline   tint   in    the 
indicator.     The  reaction,  however,  varies  with  composition   of 
the  medium,   the   important   factors    being  the  percentage  of 
sugar  and  the  reaction. 

C.  Production   of  Indol. — The    b.     coli    produces    indol    in 
peptone  water.     The  methods  have  been  given  on  page  82,  and 
for  the  detection  of  the  reaction  the  use  of  Ehrlich's  rosindol 
test  is  preferable ;  (if  the  nitroso-indol  reaction  be  used,  a  small 
quantity  of   a   nitrite   must   be   added).     Two   peptone   tubes 
should  always  l>e  inoculated,  and  if  the  reaction  is  not  obtainable 
in  one  after    two  or   three  days'  growth,  the  other  should  be 
incubated  for  from  six  to  seven  days  and  then  tested.     Where  a 
faint  reaction  is  obtained,  it  is  well  to  corroborate  the  presence  of 
indol  by  dissolving  the  rosindol  out  with  amyl-alcohol  as  described. 

23 


354  TYPHOID  FEVER 

D.  Reduction  of  Nitrates. — The  b.  coli  is  frequently  capable 
of  reducing  nitrates  to  nitrites.  For  this  test,  Savage  recom- 
mends the  use  of  a  medium  made  by  dissolving  10  grm.  of 
peptone  in  1  litre  of  ammonia-free  distilled  water,  and  adding 
2  grm.  of  nitrite-free  potassium  nitrate.  The  medium  is  filtered, 
tubed,  and  sterilised  for  half  an  hour  on  three  days.  Tubes 
are  infected  and  incubated  for  forty-eight  hours,  the  forma- 
tion of  nitrites  being  now  tested  for  by  Ilosvay's  method. 
The  following  solutions  are  required  :  (a)  sulphanilic  acid,  -5 
grm.  dissolved  in  150  c.c.  dilute  acetic  acid  (s.g.  1'04);  (6) 
1  grm.  a-naphthylamine  is  dissolved  in  22  c.c.  of  water,  the 
solution  filtered,  and  180  c.c.  dilute  acetic  acid  added.  In 
using  the  test,  2  c.c.  of  each  of  these  solutions  is  added  to  10  c.c. 
of  culture.  If  reduction  of  the  nitrates  has  occurred,  a  rose 
pink  colour  should  develop  almost  immediately.  It  is  to  be 
noted  that  the  pink  colour  first  produced  sometimes  disappears 
as  it  is  formed  or  on  shaking ;  in  such  a  case  further  portions 
of  the  two  reagents  in  equal  quantities  should  be  added. 

Agglutination  Eeactions  of  the  B.  coli. — When  the  b.  coli  has 
produced  a  pathological  condition  in  an  animal,  the  serum  of 
the  infected  animal  frequently  manifests  specific  agglutinative 
characters,  especially  towards  the  strain  of  the  organism  isolated 
from  the  lesions.  Under  certain  circumstances,  also,  the  serum 
of  an  animal  infected  by  some  other  member  of  the  b.  coli  group 
may  also  agglutinate  strains  of  this  organism.  This  subject  will 
be  treated  of  when  we  consider  the  differentiation  of  the 
members  of  the  group  one  from  another. 

Isolation  of  the  B.  coli. — In  the  case  of  abscesses  or  coli 
infection  of  the  kidney  or  bladder,  etc.  (p.  356),  the  isolation  of 
the  organism  is  usually  easy,  the  use  of  agar  plates  being  here 
sufficient.  When,  however,  the  organism  is  present  along  with 
other  bacteria,  as  in  the  case  of  water,  sewage,  etc.,  special  means 
must  be  adopted,  the  object  of  which  usually  is  to  inhibit  the 
growth  of  all  organisms  except  those  belonging  to  the  coli  group. 
Formerly  media  containing  small  quantities  of  carbolic  acid 
were  used  for  this  purpose,  but  now  the  inhibition  is  usually 
effected  by  the  use  of  certain  aniline  dyes,  by  picric  acid,  or 
by  bile  salts.  The  media  of  Conradi-Drigalski,  Conradi,  Endo, 
Fawcus,  'and  of  MacConkey  (pp.  47-51)  are  examples.  All 
these  media  have  their  uses,  and  it  is  best  to  select  that  with 
which  the  worker  has  had  most  experience.  In  this  country 
MacConkey 's  bile- salt  lactose  agar  is  perhaps  most  widely  used. 
The  methods  of  the  application  of  these  media  and  the  appear- 
ances of  b.  coli  have  already  been  described  (p.  47-51). 


THE  RECOGNITION  OF  TYPICAL  B.  COLI      355 

The  Recognition  of  typical  B.  coli. — The  work  on  b.  coli, 
especially  in  relation  to  its  occurrence  in  water,  has  revealed 
the  existence  of  a  very  large  number  of  varieties  of  the  organism. 
These  differ  from  one  another  in  the  absence  of  one  or  more 
of  the  characters  which  may  be  elucidated  by  the  application 
of  the  biological  methods  given.  Considerable  difference  of 
opinion  exists  as  to  what  characters  are  to  be  looked  upon  as 
type  characters,  i.e.  characters  shared  by  the  greatest  number  of 
varieties  isolated.  In  this  connection  it  is  to  be  noted  that  as 
the  b.  coli  was  originally  isolated  from  the  human  intestine,  and 
as  the  detection  of  such  intestinal  bacteria  outside  the  body 
constitutes  a  most  important  practical  question,  the  inquiry  for 
type  characters  is  to  a  certain  extent  limited  to  an  attempt  to 
arrive  at  the  type  most  frequently  present  in  the  human  intestine. 

Two  standards  may  be  alluded  to.  First,  that  of  an  English 
Committee  which  reported  in  1904  on  the  standardisation  of 
methods  for  the  bacterioscopic  examination  of  water.  According 
to  this,  the  b.  coli  is  a  small,  motile,  non-sporing  bacillus,  capable 
of  growing  at  37°  C.,  decolorised  by  Gram,  never  liquefying 
gelatin,  producing  clot  and  permanent  acidity  of  milk  within 
seven  days  at  37°,  fermenting  glucose  and  lactose,  with,  in  both, 
acid  and  gas  formation, — subsidiary  points  being  the  forma- 
tion of  indol,  the  formation  of  a  thick  yellowish-brown  growth 
on  potato,  production  of  fluorescence  in  neutral-red,  reduction  of 
nitrates,  and  fermentation  of  saccharose.  A  similar  American 
Committee  looked  upon  the  typical  organism  as  a  non-sporing 
bacillus,  motile,  fermenting  dextrose-broth,  with  the  formation 
of  about  50  per  cent,  of  gas,  of  which  about  one-third  is  carbon 
dioxide,  causing  acid  and  clot  in  milk  in  forty-eight  hours,  not 
liquefying  gelatin,  producing  indol  and  reducing  nitrates. 
These  two  standards  differ  in  the  fact  that  the  English  Committee 
lay  less  weight  on  indol  formation  and  the  reduction  of  nitrates. 

Generally  speaking,  the  application  in  any  case  of  the  scheme 
of  the  English  Committee  is  to  be  recommended,  and  organisms 
conforming  to  the  tests  laid  down  may  be  accepted  in  the 
majority  of  cases  as  probably  having  come  from  an  intestinal 
source.  The  further  differentiation  of  organisms  conforming  to 
this  type  will  be  dealt  with  later  (p.  391).  Meantime  it  may  be 
said  that,  in  addition  to  the  type  characters,  lactose-fermenters 
from  the  human  intestine  usually  ferment  saccharose  and  dulcite 
and  have  no  effect  on  adonite,  inulin  and  inosite,  and  it  may  be, 
no  influence  on  mannite. 

Pathogenic  Properties  of  the  B.  coli. — In  man,  the  b. 
coli  has  been  found  as  the  only  organism  present  in  various 


356  TYPHOID  FEVER 

suppurative  conditions  (see  Chapter ..  VII.),  especially  in  con- 
nection with  the  intestine  (e.g.  appendicitis)  and  about  the 
urinary  tract.  In  the  latter,  it  is  also  responsible  for  catarrhal 
conditions  in  the  pelvis  of  the  kidney  and  in  the  bladder,  these 
being  more  common  in  the  female,  and  frequently  presenting 
chronic  characters.  As  a  practical  point,  it  may  be  said  that 
the  treatment  of  the  latter  by  vaccines,  especially  when  made 
from  the  strain  isolated  from  the  lesion,  has  been  attended  with 
marked  success.  The  b.  coli  is  also  apparently  the  cause  of 
some  cases  of  summer  diarrhoea  (cholera  nostras),  of  infantile 
diarrhoea,  and  of  some  food  poisonings. 

The  Pathogenicity  of  the  B.  coli  and  its  Relation  to  that  of  the 
Typhoid  Bacillus. — Intraperitoneal  injection  in  guinea-pigs  is  often 
fatal.  Subcutaneous  injection  may  result  in  local  abscesses,  and  some- 
times in  death  from  cachexia.  Sanarelli  found  that  the  b.  coli  isolated 
from  typhoid  stools  was  much  more  virulent  than  when  isolated  from 
the  stools  of  healthy  persons.  He  holds  that  the  increase  in  virulence  is 
due  to  the  effect  of  typhoid  toxins.  This  increased  virulence  of  the 
b.  coli  in  the  typhoid  intestine  makes  it  possible  that  some  of  the  patho- 
logical changes  in  typhoid  may  be  due,  not  to  the  typhoid  bacillus,  but 
to  the  b.  coli.  Some  of  the  general  symptoms  may  be  intensified  by 
the  absorption  of  toxic  products  formed  by  it  and  by  other  organisms. 
Differences  of  behaviour  of  the  two  bacilli  in  connection  with  their 
pathological  effects  have  been  brought  forward  as  confirmatory  of  the 
fact  of  their  being  distinct  species.  Thus  Sanarelli  accustomed  the 
intestinal  mucous  membrane  of  guinea-pigs  to  toxins  derived  from  an 
old  culture  of  the  b.  coli,  by  introducing  day  by  day  small  quantities 
of  the  latter  into  the  stomach.  When  a  relatively  large  dose  could  be 
tolerated,  it  was  found  that  the  introduction  in  the  same  way  of  a  small 
quantity  of  typhoid  toxin  was  still  followed  by  fatal  result.  Pfeiffer  also 
found  that  while  the  serum  of  convalescents  from  typhoid  paralysed  the 
typhoid  bacilli,  it  had  no  more  effect  on  similar  numbers  of  b.  coli  than 
the  serum  of  healthy  men. 


THE  BACILLUS  TYPHOSUS. 

Bacillus  Typhosus. — Microscopic  Appearances. — It  is  some- 
times difficult  to  find  the  typhoid  bacilli  in  the  organs  of  a 
typhoid  patient.  Numerous  sections  of  different  parts  of  a 
spleen,  for  example,  may  be  examined  before  a  characteristic 
group  is  found.  The  best  tissues  for  examination  are  a  Peyer's 
patch  where  ulceration  has  not  yet  commenced  or  where  it  is 
just  commencing,  the  spleen,  the  liver,  or  a  mesenteric  gland. 
The  spleen  and  liver  are  better  than  the  other  tissues  named,  as 
in  the  latter  the  presence  of  the  b.  coli  is  more  frequent.  From 
scrapings  of  such  solid  organs  dried  films  may  be  prepared  and 
stained  for  a  few  minutes  in  the  cold  by  any  of  the  strong 


APPEARANCES  OF  CULTURES 


357 


>taining  solutions,  e.g.  with  carbol-thionin-blue,  or  with  Ziehl- 
Xerlsen  carbol-fuchsin  diluted  with  five  parts  of  distilled  wrater. 
As  a  rule,  decolorising  is  not  necessary.  For  the  proper 
observation  of  the  arrangement  of  the  bacilli  in  the  tissues, 
paratlin  sections  should  be  stained  in  carbol-thionin-blue  for  a 
few  minutes,  or  in  Loftier'*  methylene-blue  for  one  or  two  hours. 
The  bacilli  take  up  the  stain  somewhat  slowly,  and  as  they  are 
also  easily  decolorised,  the  aniline-oil  method  of  dehydration 
may  be  used  with  ad- 
vantage (vide  p.  100). 
In  such  preparations  the 
characteristic  appearance 
to  IK-  looked  for  is  tilt- 
occurrence  of  groups  of 
bacilli  lying  between  the 
cells  of  the  tissue  (Fig. 
106).  The  individual 
bacilli  are  2  p  to  4  //. 
Ion;:,  with  somewhat 
oval  ends,  and  '5  \L  in 
thickness.  Sometimes 
filaments  8  JJL  to  IQ  p 
long  may  be  observed, 
though  they  are  less 
common  than  in  cultures. 
It]  is  evident  that  one 
of  the  bacilli  may  fre- 
quently in  a  section  be 
viewed  endwise,  in  which 
case  the  appearance  will 
be  circular.  This  ap- 
pearance accounts  for  some,  at  least,  of  the  coccus-like  forms 
which  have  been  described.  The  bacilli  are  decolorised  by 
Grant 'j  method. 

Isolation  and  Appearances  of  Cultures.— To  grow  the 
organism  artificially  it  is  best  to  isolate  it  from  the  spleen  (for 
method,  see  p.  146)  as  it  exists  there  in  greater  numbers  than  in 
the  other  solid  organs,  and  may  be  the  sole  organism  present 
even  some  time  after  death.  Agar  or  gelatin  plates  or  agar  stroke 
cultures  may  be  employed.  On  the  agar  media  the  growths  are 
\Mble  after  twenty-four  hours'  incubation  at  37°  C.  On  agar 
plates  the  superficial  colonies  are  thin  and  film-like,  circular  or 
slightly  irregular  at  the  margins,  dull  white  by  reflected  light, 
bluish-grey  by  transmitted  light.  Colonies  in  the  substance  of 


•Fie.  106. — A  large  clump  of  typhoid  bacilli 
in  a  spleen.  The  individual  bacilli  are  only 
seen  at  the  periphery  of  the  mass.  (In 
this  spleen  enormous  numbers  of  typhoid 
bacilli  were  shown  by  cultures  to  be  present 
in  a  practically  pure  condition.) 

Paraffin  section  ;  stained  with  carbol-thionin 
blue,  x  500. 


358 


TYPHOID  FEVER 


the  agar  are  small,  and  appear  as  minute  round  points.  Under  a 
low  objective,  the  surface  colonies  are  found  to  be  very  transparent 
(requiring  a  small  diaphragm  for  their  definition),  finely  granular 
in  appearance,  and  with  a  very  coarsely  crenated  and  well- 
defined  margin.  The  deep  colonies  are  usually  spherical,  some- 
times lenticular  in  shape,  and  are  smooth  or  finely  granular  on 
the  surface,  and  more  opaque  than  the  superficial  colonies.  In 
cover-glass  preparations,  the  bacilli  are  found  to  present  the  same 
microscopic  appearances  as  in  preparations  from  solid  organs, 
except  that  there  may  be  a  greater  number  of  the  longer  forms 
which  may  almost  be  called  filaments  (Fig.  107).  The  same  is 

true  of  films  made  from 
young  gelatin  cultures. 
Sometimes  the  diversity 
in  the  length  of  the 
bacilli  is  such  as  to  throw 
doubt  on  the  purity  of 
the  culture.  As  a  general 
rule,  in  a  young  (twenty - 
four  or  forty-eight  hours 
old)  culture,  grown  at  a 
uniform  temperature,  the 
bacilli  are  plump,  and 
the  protoplasm  stains 
uniformly.  In  old  cul- 
tures, or  in  cultures 
which  have  been  exposed 
to  changes  of  tempera- 
ture, the  protoplasm 
stains  only  in  parts ; 
there  may  be  an  appear- 
ance of  irregular  vacuolation  either  at  the  centre  or  at  the  ends 
of  the  bacilli. 

Motility. — In  hanging-drop  preparations  the  bacilli  are  found 
to  be  actively  motile.  The  smaller  forms  have  a  darting  or 
rolling  motion,  passing  quickly  across  the  field,  whilst  some  show 
rapid  rotatory  motion.  The  filamentous  forms  have  an  undu- 
lating or  serpentine  motion,  and  move  more  slowly.  Hanging- 
drop  preparations  ought  to  be  made  from  agar  or  broth  cultures 
not  more  than  twenty-four  hours  old.  In  older  cultures  the 
movements  are  less  active. 

Flagella. — On  being  stained  by  the  appropriate  methods 
(vide  p.  110),  the  bacilli  are  seen  to  possess  many  long  wavy 
fiagella  which  are  attached  all  along  the  sides  and  to  the  ends 


FIG.  107.— Typhoid  bacilli,  from  a  young 
culture  on  agar,  showing  some  filamentous 
forms. 

Stained  with  weak  carbol-fuschin.      x  1000. 


APPEARANCES  OF  CULTUKKs 


359 


(!•'!::.    108).     They  are  more  numerous,  longer,  and  more  wavy 
than  those  of  the  b.  coli. 

Characters  of  Culture. — Generally  speaking,  on  artificial 
media  growths  of  the  b.  typhosus  appear  less  dense  than 
those  of  the  b.  coli.  Stab  cultures  in  peptone  gelatin  give 
a  somewhat  characteristic  appearance.  On  the  surface  of  the 
medium  growth  spreads  outwards  from  the  puncture  as  a  thin 
leaf-like  film  or  pellicle,  with  irregularly  wavy  margin  (Fig. 


FIG.  108. — Typhoid  bacilli,  from  a  young  culture  on  agar,  showing 

Hagella.     See  also  Plate  III.,  Fig.  20. 
Stained  l>y  Van  Ennengem's  method,      x  1000. 

109,  A).  It  is  semi-transparent  and  of  bluish-white  colour. 
Ultimately  this  surface  growth  may  reach  the  wall  of  the  tube. 
Not  infrequently,  however,  the  surface  growth  is  not  well 
marked.  Along  the  stab  there  is  an  opaque  whitish  line  of 
growth,  of  finely  nodose  appearance.  There  is  no  liquefaction 
of  the  medium,  and  no  formation  of  gas.  In  stroke  cultures 
there  is  a  thin  bluish-white  film,  but  it  does  not  spread  to  such 
an  extent  as  in  the  case  of  the  surface  growth  of  a  stab  culture 
(Fig.  109,  B).  In  gelatin  plates  also  the  superficial  and  deep 


360 


TYPHOID  FEVER 


colonies  present  corresponding  differences.  The  former  are 
delicate  semi-transparent  films,  with  wavy  margin,  and  are 
much  larger  than  the  colonies  in  the  substance,  which  appear  as 
small  round  points  (Fig.  110).  These  appearances,  which  are 
well  seen  on  the  third  or  fourth  day,  resemble  those  seen  in  agar 
plates,  as  already  described  in  the  method  of  isolation ;  but  on 
gelatin  the  surface  colonies  are  rather  more  transparent  than 


FIG.  109. 

A.  Stab  culture  of  the  typhoid  bacillus  in  gelatin,  five  days'  growth. 

B.  Stroke  culture  of  the  typhoid  bacillus  on  gelatin,  six  days  growth. 

C.  Stab  culture  of  the  bacillus  coli  in  gelatin,  nine  days'  growth";  the  gelatin 

is  split  in  its  lower  part  owing  to  the  formation  of  yas. 


those  on  agar.  Their  characters,  as  seen  under  a  low  power 
of  the  microscope,  also  correspond.  If  a  gelatin  tube  be 
inoculated  and  incubated  at  37°  C.,  a  uniform  turbidity  is 
produced  (cf.  b.  coli,  p.  351). 

In  stroke  cultures  on  agar  there  is  a  bluish-grey  film  of 
growth,  with  fairly  regular  margins,  but  without  any  character- 
istic features.  This  film  is  loosely  attached  to  the  surface,  and 
can  be  easily  scraped  off. 


BIOLOGICAL  REACTIONS  361 

K  The  growth  on  potatoes  is  important.  For  several  days  (at 
incubation  temperature)  after  inoculation  there  is  apparently  no 
growth.  If  looked  at  obliquely,  the  surface  appears  wet,  and  if 
it  is  scraped  with  the  platinum  loop,  a  glistening  track  is  left ;  a 
cover-glass  preparation  shows  numerous  bacilli.  Later,  however, 
a  slight  pellicle  with  a  dull,  somewhat  velvety  surface  may 
appear,  and  this  may  even  assume  a  brown  appearance.  These 
characteristic  appearances  are  only  seen  when  a  fresh  potato 
with  an  acid  reaction  lias 
been  used. 

In  bouillon  incubated 
at  37°  C.  for  twenty-four 
hours  there  is  simply 
a  uniform  turbidity. 
Cover-glass  preparations 
made  from  such  some- 
times show  filamentous 
forms  of  considerable 
length  without  apparent 
segmentation. 

CoHf/ifions   of  (,'rntrtl,, 

ttc. — The  optimum  tem- 

I't-nit  mv  of  the  typhoid  "^^BBBB^^" 

bacillus  is  about  37°  C., 

tlmntrh   it  flkn  flourish^*     FlG-  HO.— Colonies  of  the  typhoid  bacillus 

(one  superficial  and  three  deep)  in  a  gelatin 

well    at    the    room    tern-        plate.    Three  days'  growth  at  room  tem- 
perature.       It     will     not         perature.     x!5. 
grow    below    9°    C.    or 

above  42°  C.  Its  powers  of  resistance  correspond  with  those 
of  most  non-sporing  bacteria.  It  is  killed  by  exposure  for 
half  an  hour  at  60°  C.,  or  for  two  or  three  minutes  at  100°  C. 
Typhoid  bacilli  kept  in  distilled  or  in  ordinary  tap  water  have 
usually  been  found  to  be  dead  after  three  weeks  (Frankland). 

Biological  Reactions.  —  Very  important  means  of  identi- 
fying the  typhoid  bacillus  are  found  in  testing  its  capa- 
cities for  growth  on  certain  special  media.  This  facilitates 
its  being  differentiated  from  the  b.  coli  and  the  other 
members  of  the  coli-typhoid  group.  The  following  results  will 
be  best  appreciated  if  considered  in  relation  to  what  is  said 
regarding  these  other  organisms,  as  the  reactions  of  the  typhoid 
bacilli  in  differentiating  media  are  largely  negative.  (See 
Table,  p.  394.) 

The  testa  with  sugars  are  important.  The  typhoid  bacillus 
produces  acid  without  gas  in  maltose,  laevulose,  glucose,  and 


362  TYPHOID  FEVER 

mannite,  but  originates  no  change  in  lactose,  cane-sugar,  or 
dulcite.  Further,  no  gas  production  is  observed  in  gelatin 
shake  cultures,  and  there  is  no  curdling  of  milk,  although  in 
litmus  milk  slight  acid  production  occurs ;  in  a  time  varying 
from  a  few  days  to  a  month  the  acid  change  may  be  succeeded 
by  alkali  production.  Under  ordinary  circumstances,  the 
typhoid  bacillus  is  incapable  of  producing  indol  in  peptone-salt 
solution,  and  does  not  alter  neutral-red  in  lactose  bouillon. 

A  great  many  special  tests  were  formerly  in  use  for  differ- 
entiating the  b.  typhosus  from  the  b.  coli.  The  use  of  these 
is  not  now  so  necessary,  but  the  following  may  be  described  : — 

The  Media  of  Capaldi  and  Proskauer.—The  first  of  these  ("No.  1  ")  is 
a  medium  free  of  albumin,  in  which  b.  coli  grows  well  and  freely  produces 
acid,  while  the  typhoid  bacillus  hardly  grows  at  all,  and  certainly  will 
produce  no  change  in  the  reaction.  Its  composition  is  as  follows  : 
asparagin  '2  parts,  mannite  '2,  sodium  chloride  '02,  magnesium  sulphate 
'01,  calcium  chloride  '02,  potassium  monophosphate  '2,  distilled  water 
to  100  parts.  The  second  medium  ("No.  2")  contains  albumin,  and 
in  it  the  b.  coli  produces  no  acid,  while  the  typhoid  bacillus  grows  well 
and  produces  an  acid  reaction.  It  consists  of  Witte's  peptone  2  parts, 
mannite  '1,  distilled  water  to  100  parts.  After  the  constituents  of  each 
medium  are  mixed  and  dissolved,  it  is  steamed  for  one  and  a  half  hours 
and  then  made  neutral  to  litmus — the  first  medium,  being  usually 
naturally  acid,  by  sodium  hydrate,  the  second,  being  usually  alkaline,  by 
citric  acid.  The  medium  is  then  filtered,  filled  into  tubes  containing 
5  c.c.,  and  these  are  sterilised.  After  inoculation  for  twenty  hours  the 
reaction  of  the  medium  is  tested  by  adding  litmus. 

The  identification  of  the  typhoid  bacillus  is  best  facilitated 
by  means  of  agglutination  reactions  which  will  be  treated  of 
later  (pp.  371,  393). 

Pathological  Changes  in  Typhoid  Fever. — Here  wre  confine 
our  attention  solely  to  the  bacteriological  aspects  of  the  disease. 
The  inflammation  and  ulceration  in  the  Peyers  patches  and 
solitary  glands  of  the  intestine  are  the  central  features.  In  the 
early  stage  there  is  produced  an  acute  inflammatory  condition, 
attended  with  extensive  leucocytic  emigration  and  sometimes 
with  small  haemorrhages.  At  this  period  the  typhoid  bacilli  are 
most  numerous  in  the  patches,  groups  being  easily  found  between 
the  cells.  The  subsequent  necrosis  is  evidently  in  chief  part 
the  result  of  the  action  of  the  toxic  products  of  the  bacilli, 
which  gradually  disappear  from-  their  former  positions,  though 
they  may  still  be  found  in  the  deeper  tissues  and  at  the  spread- 
ing margin  of  the  necrosed  area.  They  also  occur  in  the  lym- 
phatic spaces  of  the  muscular  coat.  It  is  to  be  remarked  that 
the  number  of  the  ulcers  arising  in  the  course  of  a  case  bears 


PATHOLOGICAL  CHANGES  363 

no  relation  to  its  severity.  Small  ulcers  may  occur  in  the 
Ivmphoid  follicles  of  the  large  intestine. 

The  me&enteric  glands  corresponding  to  the  affected  part  of 
the  intestine  are  usually  enlarged,  sometimes  to  a  very  great 
extent,  the  whole  mesentery  being  filled  with  glandular  masses. 
In  such  glands  there  may  be  acute  inflammation,  and  occasion- 
ally necrosis  in  patches  occurs.  Sometimes  on  section  the 
glands  are  of  a  pale-yellowish  colour,  the  contents  being  diffluent 
and  consisting  largely  of  leucocytes.  Typhoid  bacilli  may  be 
isolated  both  from  the  glands  and  the  lymphatics  connected  with 
them,  but  the  b.  coli  is  in  addition  often  present. 

The  spleen  is  enlarged, — on  section  usually  of  a  fairly  firm 
consistence,  of  a  reddish-pink  colour,  and  in  a  state  of  conges- 
tion. Of  all  the  solid  organs  it  usually  contains  the  bacilli  in 
greatest  numbers.  They  can  be  seen  in  sections,  occurring  in 
clumps  between  the  cells,  there  being  no  evidence  of  local 
reaction  round  them  (Fig.  106).  Similar  clumps  may  occur  in 
the  liver  in  any  situation,  and  without  any  local  reaction.  In 
this  organ,  however,  there  are  often  small  foci  of  leucocytic 
infiltration,  in  which,  so  far  as  our  experience  goes,  bacilli 
cannot  be  demonstrated.  The  bacillus  is  found,  often  in  large 
numbers,  in  the  gall-bladder,  where  it  may  persist  for  years 
(vide  infra).  Clumps  of  bacilli  may  also  occur  in  the  kidney. 

In  addition  to  these  local  changes  in  the  solid  organs,  there  are  also 
\\  i(lc>j)ic;i(l  cellular  degenerations  in  the  solid  organs  which  suggest  the 
action  of  toxic  products. 

In  the  lunys  there  may  be  bronchitis,  patches  of  congestion  and  of 
acute  broncho-pneumonia.  In  these,  typhoid  bacilli  may  sometimes  be 
observed,  but  evidence  of  a  toxic  action  depressing  the  powers  of  resist- 
ance of  the  lung  tissue  is  found  in  the  fact  that  the  pneumococcus 
frequently  occurs  in  such  complications  of  typhoid  fever. 

The  nervous  system  shows  little  change,  though  meningitis  associated 
either  with  the  typhoid  bacillus,  with  the  b.  coli,  or  with  the  strepto- 
coccus pyogenes  has  been  observed. 

In  typhoid  fever  the  bacilli  can  in  90  per  cent,  of  cases  be  isolated  from 
the  blood  during  the  course  of  the  illness.  The  local  lesions  are  thus  associ- 
ated with  a  general  septica.jmic  process.  The  bacilli  have  been  found  in 
the  rofteolar  spots  which  occur  in  typhoid  fever,  but  it  cannot  he  yet 
stated  that  such  spots  are  always  due  to  the  presence  of  the  bacilli.  The 
fact  that  the  typhoid  bacilli  are  usually  confined  to  certain  organs  and 
tissues  shows  that  they  probably  have  a  selective  action  on  certain  tissues. 

To  sum  up  the  pathology  of  typhoid  fever,  we  have  in  it  a 
disease  the  centre  of  which  lies  in  the  lymphoid  tissue  in  and 
connected  with  the  intestine.  In  this  situation  we  must  have 
an  irritant,  against  which  the  inflammatory  reaction  is  set  up, 


364  TYPHOID  FEVER 

and  which  in  the  intestine  is  sufficiently  powerful  to  cause 
necrosis.  The  affections  of  the  other  organs  of  the  body  suggest 
the  circulation  in  the  blood  of  poisonous  substances  capable  of 
depressing  cellular  vitality,  and  producing  histological  changes. 
The  occurrence  of  bacilli  in  the  blood  and  organs  links  typhoid 
fever  with  septicsemic  processes. 

Suppuration  occurring  in  connection  with  Typhoid  Fever.— 
With  regard  to  the  relation  of  the  typhoid  bacillus  to  such 
conditions,  statements  as  to  its  isolation  from  pus,  etc.,  can  be 
accepted  only  when  all  the  points  available  for  the  diagnosis  of 
the  organism  have  been  attended  to.  On  this  understanding  the 
following  summary  may  be  given  : — In  a  certain  proportion  of 
the  cases  examined  the  typhoid  bacillus  has  been  the  only  organ- 
ism found.  This  has  been  the  case  in  subcutaneous  abscesses, 
in  suppurative  periostitis,  suppuration  in  the  parotid,  abscesses 
in  the  kidneys,  etc.,  and  probably  also  in  one  or  two  cases  of 
ulcerative  endocarditis.  But  in  the  majority  of  cases  other 
organisms,  especially  the  b.  coli  and  the  pyogenic  micrococci, 
have  been  obtained,  the  typhoid  bacillus  having  been  searched  for 
in  vain.  It  has,  moreover,  been  experimentally  shown,  notably 
by  Dmochowski  and  Janowski,  that  suppuration  can  be  experi- 
mentally produced  by  injection  in  animals,  especially  in  rabbits, 
of  pure  cultures  of  the  typhoid  bacillus,  the  occurrence  of  sup- 
puration being  favoured  by  conditions  of  depressed  vitality,  etc. 
These  observers  also  found  that  when  typhoid  bacilli  were 
injected  along  with  pyogenic  staphylococci,  the  former  died  out 
in  the  pus  more  quickly  than  the  latter.  Accordingly,  in  clinical 
cases  where  the  typhoid  bacillus  is  present  alone,  it  is  improbable 
that  other  organisms  were  present  at  an  earlier  date. 

Occurrence  of  Gallstones  in  those  who  have  suffered  from 
Typhoid  Fever. — As  has  been  stated,  foci  of  bacilli  occur  in  the 
liver  in  typhoid  fever,  and  these  bacilli  are  excreted  with  the 
bile.  In  the  gall-bladder  they  apparently  not  infrequently  set 
up  a  catarrhal  process  in  the  biliary  ducts  and  gall-bladder 
(cholecystitis  typhosa\  and  are  then  in  a  better  position  for  multi- 
plication, in  consequence  of  the  presence  of  albuminous  catarrhal 
secretions.  There  is  evidence  that  the  bacilli  may  persist  in  the 
gall-bladder  for  many  years,  and  probably  the  catarrhal  inflam- 
mation which  they  keep  up  is  responsible  for  many  of  the  cases 
of  gallstones  wrhich  occur — the  albuminous  matter  produced 
causing  a  deposit  of  the  bile  in  a  solid  form.  Typhoid  bacilli 
have  actually  been  isolated  from  cases  of  gallstones  operated  on 
years  after  an  attack  of  typhoid  fever,  and  the  bacilli  have  even 
been  found  within  the  calculi.  They  have  also  been  demon- 


PATHOGENIC  EFFECTS  OF  B.  TYPHOSUS      365 

strated  in  chronic  suppurations  occurring  in  the  gall-bladder. 
It  is  to  be  noted  that  gallstones  are  more  frequently  found  in 
women  than  in  men,  the  proportion  being  about  four  to  one, 
and  probably  a  considerable  proportion  of  the  total  number  of 
cases  of  gallstones  are  traceable  to  the  previous  occurrence  of 
typhoid  fever. 

Pathogenic  Effects  produced  in  Animals  by  the  Typhoid 
Bacillus. — There  is  no  disease  of  animals  which  can  be  said 
to  be  identical  with  typhoid,  nor  is  there  any  evidence  of  the 
occurrence  of  the  typhoid  bacillus  under  ordinary  pathological 
conditions  in  the  bodies  of  animals.  Attempts  to  communicate 
the  disease  to  animals  by  feeding  them  on  typhoid  dejecta  have 
been  unsuccessful,  and  though  pathogenic  effects  have  been 
produced  by  introducing  pure  cultures  in  food,  the  disease  has 
usually  borne  no  resemblance  to  human  typhoid.  The  most 
successful  experiments  have  been  those  of  Remlinger,  who,  by 
continuously  feeding  rabbits  on  vegetables  soaked  in  water  con- 
taining typhoid  bacilli,  produced  in  certain  cases  symptoms 
resembling  those  of  typhoid  fever  (diarrhrea,  remittent  pyrexia, 
etc.).  An  agglutinating  action  was  observed  in  the  serum,  and 
post  mortem  there  was  congestion  of  the  Peyer's  patches,  and 
typhoid  bacilli  were  isolated  from  the  spleen. 

Feeding  experiments  are  thus  unsatisfactory,  and  the  same 
may  be  said  of  the  results  of  subcutaneous  or  intraperitoneal 
infection.  Here,  again,  pathogenic  effects  can  easily  be  produced 
by  the  typhoid  bacillus,  but  these  effects  are  of  the  nature  of  a 
short  acute  illness  characterised  by  pyrexia,  rapid  loss  of  weight, 
inability  to  take  food,  and  frequently  ending  fatally  in  from 
twenty-four  to  forty-eight  hours.  The  type  of  disease  is  thus 
very  different  from  what  occurs  naturally  in  man.  In  such 
injection  experiments  the  results  vary  considerably,  sometimes 
scarcely  any  effect  being  produced  by  a  large  dose  of  a  culture. 
This  is  no  doubt  due  to  the  fact  that  different  strains  of  the 
bacillus  vary  much  in  virulence.  Ordinary  laboratory  cultures 
are  often  almost  non-pathogenic.  They  can,  however,  be  made 
virulent  in  various  ways.  Sanarelli  used  the  method  of  injecting 
sterilised  cultures  of  the  b.  coli  intraperitoneally  at  the  same 
time  as  the  typhoid  bacillus  was  introduced  subcutaneously. 
After  this  procedure  had  been  repeated  through  a  series  of 
animals,  a  typhoid  culture  of  exalted  virulence  was  obtained. 
Sidney  Martin  has  obtained  virulent  cultures  by  passing  bacilli, 
derived  directly  from  the  spleen  of  a  person  dead  of  typhoid 
fever,  through  the  peritoneal  cavities  of  a  series  of  guinea-pigs. 

Sanarelli,  studying  the  effects  of  the  intraperitoneal  injection 


366  TYPHOID  FEVER 

of  a  few  drops  of  a  culture  of  highly  exalted  virulence,  found 
that  the  Peyer's  patches  and  solitary  glands  of  the  intestine  were 
enormously  infiltrated,  sometimes  almost  purulent,  and  that  they 
contained  typhoid  bacilli,  as  also  did  the  mesenteric  lymphatics 
and  glands,  and  the  spleen.  These  results  are  interesting,  but 
have  not  been  confirmed. 

The  Toxic  Products  of  the  Typhoid  Bacillus. — Here  very 
little  light  has  been  thrown  on  the  pathology  of  the  disease,  but 
the  general  results  may  be  outlined.  We  may  state  that  there 
exist  in  the  bodies  of  typhoid  bacilli  toxic  substances,  that  in 
artificial  cultures  these  do  not  pass  to  any  great  degree  out  into 
the  surrounding  medium,  and  that  though  they  produce  effects 
on  the  intestine,  there  is  evidence  that  such  effects  are  not 
characteristic  and  not  peculiar  to  the  toxins  of  the  b.  typhosus. 
Sidney  Martin  found  that  the  bodies  of  bacteria  killed  by  chloro- 
form vapour  were  very  toxic, — more  so  than  filtered  cultures. 
Diarrhoea  was  a  constant  symptom  after  injection,  but  no  change 
in  the  Peyerian  patches  was  observed.  He  also  found  that 
virulent  cultures  of  the  b.  coli  gave  similar  results  when 
similarly  treated.  Allan  Macfadyen,  by  grinding  up  typhoid 
bacilli  frozen  solid  by  liquid  air,  produced  a  fluid  whose  toxic 
effect  he  attributed  to  the  presence  of  the  intracellular  poisons. 

The  Immunisation  of  Animals  against  the  Typhoid 
Bacillus. — Earlier  observers  had  been  successful  in  accustoming 
mice  to  the  typhoid  bacillus  by  the  successive  injections  of 
small*  and  gradually  increasing  doses  of  living  cultures  of  the 
bacillus.  Later,  Brieger,  Kitasato,  and  Wassermann  found  that 
the  bacillus  when  modified  by  being  grown  in  a  bouillon  made 
from  an  extract  of  the  thymus  gland  no  longer  killed  mice  and 
guinea-pigs.  These  animals  after  injection  were  moreover 
immune,  and  it  was  also  found  that  the  serum  of  a  guinea- 
pig  thus  immunised  could,  if  transferred  to  another  guinea-pig, 
protect  the  latter  from  the  subsequent  injection  of  a  dose  of 
typhoid  bacilli  to  which  it  would  naturally  succumb.  Chante- 
messe  and  Widal,  Sanarelli,  and  also  Pfeiffer,  succeeded  in 
immunising  guinea-pigs  against  the  subsequent  intraperitoneal 
injection  of  virulent  living  typhoid  bacilli,  by  repeated  and 
gradually  increasing  intraperitoneal  or  subcutaneous  doses  of 
dead  typhoid  cultures  in  bouillon.  Experiments  performed  with 
serum  derived  from  typhoid  patients  and  convalescents  indicate 
that  similar  effects  occur  in  those  who  have  successfully  resisted 
the  natural  disease.  The  serum  of  such  patients  has  antibacterial 
powers,  but  there  is  no  evidence  that  it  contains  any  antitoxic 
bodies  (see  chapter  on  Immunity).  Pfeiffer,  for  example,  found, 


RELATIONSHIP  TO  TYPHOID  FEVER          367 

on  adding  serum  from  typhoid  convalescents  to  typhoid  bacilli 
killed  by  heat,  and  injecting  the  mixture  into  guinea-pigs,  that 
death  took  place  as  in  control  animals  which  had  received  these 
toxic  agents  alone.  Pfeiffer  also  found  that  by  using  the  serum 
of  immunised  goats,  he  could,  to  a  certain  extent,  protect  other 
animals  against  the  subsequent  injection  of  virulent  living 
typhoid  bacilli.  On  trying  to  use  the  agent  in  a  curative  way, 
i.e.  injecting  it  only  after  the  bacilli  had  begun  to  produce  their 
effects,  he  got  little  or  no  result. 

General  View  of  the  Kelationship  of  the  B.  typhosus  to 
Typhoid  Fever. — 1.  We  see  in  typhoid  fever  a  disease  having 
its  centre  in  and  about  the  intestine,  and  acting  secondarily  on 
many  other  parts  of  the  body.  In  the  parts  most  affected  there 
is  always  a  bacillus  present,  microscopically  resembling  other 
bacilli,  es[>ecially  the  b.  coli,  which  is  a  normal  inhabitant  of 
the  animal  intestine.  The  bacillus  can  be  isolated  from  the 
characteristic  lesions  of  the  disease  and  from  other  parts  of  the 
body  as  described,  and  further,  it  is  found  by  culture  and  serum 
reactions  to  differ  from  other  organisms.  Here  the  important 
point  is  that  a  bacillus  giving  all  the  reactions  of  the  typhoid 
bacillus  has  never  been  isolated  except  from  cases  of  typhoid 
fever,  or  under  circumstances  that  make  it  possible  for  the 
bacillus  in  question  to  have  been  derived  from  a  case  of  typhoid 
fever. 

2.  A  difficulty  in  the  way  of  accepting  the  etiological  relation- 
ship  of  the   b.    typhosus   lies   in   the    comparative   failure   of 
attempts    to   cause   the  disease   in   animals.     We  have  noted, 
however,    that  in  nature    animals  do  not  suffer  from  typhoid 
fever. 

3.  The  observations  of  Pfeiffer  and  others  on  the  protective 
power  against  typhoid  bacilli  shown,  on  testing  in  animals,  to 
belong  to  the  serum  of  typhoid  patients  and  convalescents,  and 
the  peculiar  action  of  such  serum  in  immobilising  and  causing 
clumping  of  the  bacilli  (vide  infra),  are  also  of  great  importance 
as  indicating  an  etiological  relationship  between  the  bacillus  and 
the  disease.     Additional  important  evidence  is  found  in  the  fact 
that  vaccination  by  means  of  the  dead  bacilli  (vide  infra)  has 
a  marked  effect  in   preventing  the  disease  from  arising   in   a 
population   exposed   to    infection,    and    also    in   lowering   the 
mortality  when  the  fever  attacks  those  who  have  been  inoculated. 
These  facts  may  thus  be  accepted  as  indirect  but  practically 
conclusive   evidence   of    the    pathogenic    relationships   of    the 
typhoid  bacillus  to  the  disease. 

According  to  our  present  results,  we  must  thus   hold  that 


368  TYPHOID  FEVER 

the  b.  typhosus  constitutes  a  distinct  species  of  bacterium,  and 
that  it  is  the  cause  of  typhoid  fever.  Evidence  of  an  important 
nature  confirmatory  of  this  view  is,  we  think,  found  in  the  fact 
that  cases  have  occurred  where  bacteriologists  have  accidentally 
infected  themselves  by  the  mouth  with  pure  cultures  of  the 
typhoid  bacillus,  and  after  the  usual  incubation  period  have 
developed  typhoid  fever.  Several  cases  of  this  kind  have  been 
brought  to  our  notice,  and  are  not,  we  think,  vitiated  by  the  fact 
that  other  similar  instances  have  occurred  without  the  subsequent 
development  of  illness.  These  latter  would  be  accounted  for  by 
a  low  degree  of  susceptibility  on  the  part  of  the  individual  or  to 
a  want  of  pathogenicity  in  the  cultures. 

As  there  is  thus  strong  evidence  of  the  etiological  relationship 
of  the  typhoid  bacillus  to  typhoid  fever,  the  view  of  the 
development  of  the  disease  usually  taken  has  been  that  the 
bacilli,  being  ingested,  multiply  in  the  intestinal  tract,  cause 
inflammation  and  necrosis  of  the  lymphoid  tissue,  and,  gaining 
an  entrance  to  the  general  circulation,  produce  the  septicsemic 
phenomena  which  we  have  described. 

Within  recent  years,  considerable  attention  has  been  attracted  to 
another  view  of  the  course  of  infection  put  forward  by  Forster  and 
his  co-workers  in  Strasburg.  According  to  this,  the  process  is  primarily 
a  septicaemia,  and  the  intestinal  manifestations  are  looked  on  as 
secondary.  The  bacilli  are  supposed  to  gain  entrance  to  the  circulation 
possibly  through  the  tonsils,  sore  throat  being  a  not  uncommon  initial 
symptom  of  typhoid  fever.  In  the  blood  they  multiply,  and,  passing 
through  the  liver,  gain  access  to  the  gall-bladder,  set  up  a  catarrhal 
inflammation  there  on  the  products  of  which  they  flourish,  and  thence 
pass  out  to  infect  the  intestine.  The  intestinal  lesions  are  either  due 
to  an  elective  action  of  bacteria  brought  by  the  blood,  or  come  from 
infection  by  the  bacilli  which  pass  out  from  the  gall-bladder, — the 
former  being  apparently  the  alternative  to  which  Forster  leans.  The 
evidence  on  which  this  view  is  based  consists,  firstly,  in  the  results 
of  animal  experiments  in  which  bacilli  introduced  intravenously  have 
been  subsequently  found  chiefly  or  solely  in  the  gall-bladder, — it  may  be, 
persisting  there  for  weeks.  Further,  it  is  stated  that  bacilli  can  be 
isolated  from  the  blood  during  the  later  parts  of  the  incubation  stage  of 
the  disease,  and  before  they  can  be  demonstrated  in  the  intestine,  where 
they  are  said  not  to  appear  until  sometime  during  the  first  week  of 
active  disease.  And  again  it  is  stated  that  in  the  bodies  of  persons 
dying  from  typhoid  fever,  while  bacilli  are  always  present  in  the  gall- 
badder  and  in  the  upper  parts  of  the  small  intestine,  they  are  frequently 
absent  from  the  lower  part  of  the  latter  and  from  the  colon.  It  cannot 
be  said  that  this  view  of  the  disease  has  been  satisfactorily  established. 
Opinion  differs  as  to  the  alleged  late  appearance  of  the  bacilli  in  the 
intestine,  and  the  infectivity  noticed  during  the  incubation  stage  must  be 
explained.  Further,  there  is  strong  reason  for  believing  that  multiplication 
of  the  bacilli  in  the  intestine  can  take  place.  The  evidence  of  this  rests 
on  the  finding  of  bacilli,  it  may  be  in  considerable  numbers,  in  the  faeces 


TYPHOID  CARRIERS  369 

and  even  in  the  blood  of  healthy  individuals  who  have  merely  been  in 
contact  with  typhoid  cases  or  typhoid  carriers,  and  who  show  no 
symptoms  of  the  disease. 

There  is  evidence  that  certain  individuals  are  relatively 
insusceptible  to  typhoid  fever.  The  cases  of  the  occurrence 
of  typhoid  bacilli  in  the  healthy  intestine  support  this  view,  and 
it  has  been  further  shown  that  during  an  epidemic  certain 
persons  may  suffer  from  slight  intestinal  symptoms  with  typhoid 
bacilli  in  the  faeces  without  the  disease  going  through  its  usual 
course.  The  so-called  "  ambulatory "  cases  of  typhoid  fever 
form  a  link  between  these  mild  infections  and  fully  developed 
typhoid  fever. 

The  Epidemiology  of  Typhoid  Fever. — Generally  speaking, 
the  former  prevalence  of  typhoid  fever  and  the  periodic  outbreaks 
which  still  occur  even  in  well-regulated  communities,  have  de- 
pended on  the  capacity  of  the  typhoid  bacillus  to  live  and  it 
may  be  to  multiply  outside  the  human  body.  The  investigation 
of  the  prevalence  of  the  typhoid  bacillus  under  such  saprophytic 
conditions  is  a  matter  of  great  difficulty,  as,  for  its  proper  study, 
the  capacity  of  the  organism  to  multiply  when  other  intestinal 
and  putrefactive  organisms  are  present  constitutes  the  essential 
problem.  Enough  is  known,  however,  to  show  that  the  bacillus 
can  remain  viable  under  such  circumstances  for  some  days,  and 
it  may  be  wreeks.  This  fact  explains  the  occurrence  of  the 
epidemics  due  to  water,  and  sometimes,  it  may  be,  to  milk 
supplies  becoming  contaminated  with  the  excreta  from  typhoid 
patients.  Where  surface  wells  are  used,  and  where  sewage, 
instead  of  being  properly  disposed  of,  finds  its  way  into  ash-heaps 
or  cesspools,  the  way  is  opened  up  for  communities  becoming 
infected  with  typhoid  fever. 

Typhoid  Carriers. — In  the  great  majority  of  cases  of  typhoid 
fever,  the  bacilli  disappear  from  the  faeces  within  from  two  to 
ten  weeks  of  convalescence,  but  in  a  certain  proportion  of  cases, 
probably  about  2  to  4  per  cent.,  evidence  is  found  of  the 
persistence  of  the  bacilli  for  many  months,  and  in  certain  cases 
their  existence  has  been  ileiiioii-tratrd  even  thirty  and,  it  may  be, 
fifty  years  after  the  attack  of  illness.  Persons  in  whom  this 
phenomenon  is  present  are  a  constant  danger  to  those  around 
them,  as  the  infectivity  of  the  bacilli  frequently  remains,  and 
during  recent  years  the  importance  of  such  "  chronic  "  carriers 
has  been  recognised  as  explaining  many  outbreaks  of  the  disease. 
The  cases  traceable  to  such  an  origin  are  of  the  type  usually 
< -lav-Til  a-  sporadic.  They  arise  amongst  persons  associated  with 
carrier-,  r-;|»eeially  when  the  latter  are  concerned  in  the  prepara- 
24 


370  TYPHOID  FEVER 

tion  of  food.  From  time  to  time,  however,  larger  epidemics 
have  arisen  from  a  carrier  having  contaminated  a  milk  supply  in 
a  dairy.  The  site  of  the  multiplication  of  the  bacteria  in  a  great 
many  of  these  carriers  is  probably  the  gall-bladder  (see  p.  364). 
As  has  been  stated,  the  typhoid  bacilli  may  persist  there  for 
many  years,  often  giving  rise  to  gallstones.  The  fact  that  women 
appear  to  be  more  liable  to  gallstones  than  men  constitutes 
a  serious  factor  in  relation  to  the  problem  of  the  typhoid  carrier, 
as  women  are  more  concerned  in  the  preparation  of  food.  An 
additional  danger  lies  in  the  fact  that  carriers  usually  appear  to 
be  in  perfect  health  or  may  only  suffer  from  slight,  and  to  them 
unimportant,  pains  in  the  region  of  the  gall-bladder,  it  being 
well  known  that  in  only  a  proportion  of  patients  suffering  from 
gallstones  do  severe  symptoms  arise.  An  additional  factor  in 
the  carrier  problem  lies  in  the  fact  stated  above,  that  apparently 
certain  persons  ingest  the  typhoid  bacilli,  and  the  latter  may 
multiply  for  some  months  in  the  intestinal  tract  without  giving 
rise  to  typhoid  fever.  Such  persons  have  been  referred  to  as 
"  paradoxical "  carriers ;  they  represent  those  who  either  are 
naturally  insusceptible  to  typhoid  fever  or  who  have  developed 
immunity  in  consequence  of  a  previous  attack ;  they  may  con- 
stitute a  danger  to  susceptible  persons  with  whom  they  may 
come  in  contact.  The  most  serious  danger  to  a  community 
arises,  however,  from  the  "  chronic  "  carrier.  In  certain  carriers, 
the  focus  of  multiplication  of  the  typhoid  bacillus  may  not  be 
the  bowel  but  the  kidney  or  bladder,  the  bacilli  in  such  cases 
passing  out  in  the  urine. 

The  tracking  down  of  'a  typhoid  carrier  constitutes  an  impor- 
tant and  difficult  problem.  Firstly,  the  serum  of  all  suspicious 
persons  ought  to  be  subjected  to  the  Widal  test  (vide  infra). 
Usually  speaking  the  carrier  gives  a  positive  reaction,  but 
sometimes  this  is  absent  and  sometimes  is  only  obtained  with  a 
low  dilution  of  the  serum.  Further,  it  has  been  shown  in 
chronic  carriers  that  the  agglutinating  capacity  of  the  serum 
varies  from  time  to  time  and  sometimes  may  be  absent.  The 
proof  of  the  presence  of  a  carrier  lies  essentially  in  the  isolation 
of  the  typhoid  bacillus  from  the  faeces  or  the  urine,  and  it  is  to 
be  noted  that,  especially  in  the  former,  the  organism  is  not 
constantly  present, — in  certain  cases  months  of  remission  have 
been  recorded.  This  of  course  may  be  due  to  the  difficulties  of 
the  search,  but  whatever  the  explanation,  it  necessitates  repeated 
examinations.  Much  work  has  been  directed  to  the  question  of 
freeing  the  typhoid  carrier  from  the  organism,  but  although 
various  methods,  such  as  intestinal  antisepsis,  vaccination, 


SERUM  DIAGNOSIS  371 

excision  of  the  gall-bladder,  have  been  tried,  success  has  hitherto 
not  been  attained.  From  the  public  health  standpoint,  the 
prevention  of  carriers  from  occurring  in  a  population  has  been 
considered,  and  it  is  a  question  whether  in  fever  hospitals 
means  ought  not  to  be  taken  for  retaining  convalescents  from 
typhoid  until  the  bodily  discharges  are  free  from  the  typhoid 
bacillus.  This  1ms  already  been  undertaken  in  the  British  army 
in  India. 

The  Serum  Diagnosis  of  Typhoid  Fever. — This  method  of 
diagnosis  is  based  on  the  fact  that  living  and  actively  motile 
typhoid  bacilli,  if  placed  in  the  diluted  serum  of  a  patient  suffer- 
ing from  typhoid  fever,  within  a  very  short  time  lose  their 
motility  and  become  aggregated  into  clumps. 

The  methods  by  which  the  test  can  be  applied  have  already 
been  described  (p.  118). 

(1)  It  will  be  there  seen  that  the  loss  of  motility  and  clumping 
may  be  observed  microscopically.     If  a  preparation  be  made  by 
the  method  detailed  (typhoid  serum  in  a  dilution  of,  say,  1  :  30 
having  been  employed),  and  examined  at  once  under  the  micro- 
scope,'the  bacilli  will  usually  be  found  actively  motile,  darting 
about  in  all  directions.     In  a  short  time,  however,  these  move- 
ments gradually  become  slower,  the  bacilli  begin  to  adhere  to  one 
another,  and  ultimately  become  completely  immobile  and  form 
clumps  by  their  aggregation.     When  this  occurs  the  reaction 
is  said  to  be  complete.     If  the  clumps  be  watched  still  longer  a 
swelling  up  of  the  bacilli  will  be  observed,  with  a  granulation 
of  the  protoplasm,  so  that  their  forms  can  with  difficulty  be 
recognised.     In  a  preparation  similarly  made  with  non-typhoid 
serum    the   individual    bacilli    can   be    observed    separate   and 
actively  motile  for  many  hours. 

(2)  A   corresponding   reaction    visible   to   the    naked  eye    is 
obtained  by  the  "  sedimentation  test,"  the  method  of  applying 
which  has  also  been  described  (p.  120).     The  test  in  this  form 
has  the  disadvantage  of  taking  longer  time  than  the  microscopic 
method,  but  it  is  useful  as  a  control ;  in  nature  it  is  similar. 

Such  is  what  occurs  in  the  case  of  a  typical  reaction.  The 
value  of  the  method  as  a  means  of  diagnosis  largely  depends 
on  attention  to  several  details.  The  race  of  typhoid  bacillus 
employed  is  important.  All  races  do  not  give  uniformly  the 
same  results,  though  it  is  not  known  on  what  this  difference  of 
susceptibility  depends.  A  race  must  therefore  be  selected 
which  gives  the  best  result  in  the  greatest  number  of  undoubted 
cases  of  typhoid  fever,  and  which  gives  as  little  reaction  a# 
with  normal  sera  or  sera  derived  from  other  diseases. 


372  TYPHOID  FEVER 

This  latter  point  is  important,  as  some  races  react  very  readily 
to  non-typhoid  sera.  Again,  care  must  be  taken  as  to  the  state  of 
the  culture  used.  The  suitability  of  a  culture  may  be  impaired 
by  varying  the  conditions  of  its  growth.  Continued  growth  of  a 
race  at  37°  C.  makes  it  less  suitable  for  use  in  the  test,  as  the 
bacilli  tend  naturally  to  adhere  in  clumps,  which  may  be 
mistaken  for  those  produced  by  the  reaction.  Wyatt  Johnson 
recommended  that  the  stock  culture  should  be  kept  growing  on 
agar  at  room  temperature  and  maintained  by  agar  sub-cultures 
made  once  a  month.  For  use  in  applying  the  test,  bouillon 
sub-cultures  are  made  and  incubated  for  twenty-four  hours  at 
37°  C.  The  relation  of  the  dilution  of  the  serum  to  the 
occurrence  of  clumping  is  most  important.  It  has  been  found 
that  if  the  degree  of  dilution  be  too  small  a  non-typhoid  serum 
may  cause  clumping.  If  possible,  observations  should  always  be 
made  with  dilutions  of  1  :  10,  1  :  30,  1  :  60,  1  :  100.  To  speak 
generally,  the  more  dilute  the  serum  the  longer  time  is  necessary 
for  a  complete  reaction.  Some  typhoid  sera  have,  however, 
very  powerful  agglutinating  properties,  and  may  in  a  compara- 
tively short  time  produce  a  reaction  when  diluted  many  hundreds 
of  times.  With  a  too  dilute  serum  not  only  may  the  reaction 
be  delayed,  but  it  may  be  incomplete, — the  clumps  formed  being 
small  and  many  bacilli  being  left  free.  These  latter  may  either 
have  been  rendered  motionless  or  they  may  still  be  motile.  No 
diagnosis  is  conclusive  which  is  founded  on  the  occurrence  of 
such  an  incomplete  clumping  alone.  Seeing  that  low  dilutions 
sometimes  give  a  reaction  with  non-typhoid  sera,  it  is  important 
to  know  what  is  the  highest  dilution  at  which  complete 
clumping  indicates  a  positive  reaction.  The  general  consensus 
of  opinion,  with  which  our  own  experience  agrees,  is  that  when 
a  serum  in  a  dilution  of  1  :  30  causes  complete  clumping  in  half 
an  hour,  it  may  safely  be  said  that  it  has  been  derived  from  a 
case  of  typhoid  fever.  Suspicion  should  be  entertained  as  to 
the  diagnosis  if  a  lower  dilution  is  required,  or  if  a  longer  time 
is  required. 

The  reaction  given  by  the  serum  in  typhoid  fever  usually 
begins  to  be  observed  about  the  seventh  day  of  the  disease, 
though  occasionally  it  has  been  found  as  early  as  the  fifth  day, 
and  sometimes  it  does  not  appear  till  the  third  week  or  later. 
Usually  it  becomes  gradually  more  marked  as  the  disease 
advances,  and  it  is  still  given  by  the  blood  of  convalescents  from 
typhoid,  but  cases  occur  in  which  it  may  permanently  disappear 
before  convalescence  sets  in.  How  long  it  lasts  after  the  end  of 
the  disease  has  not  yet  been  fully  determined,  but  in  many  cases 


SERUM  DIAGNOSIS  373 

it  has  been  found  after  several  months  or  longer.  As  a  rule,  up 
to  a  certain  point,  the  reaction  is  more  marked  where  the  fever 
is  of  a  pronounced  character,  whilst  in  the  milder  cases  it  is  less 
pronounced.  In  certain  grave  cases,  however,  the  reaction  has 
been  found  to  be  feeble  or  almost  absent.  In  some  cases,  which 
from  the  clinical  symptoms  were  almost  certainly  typhoid,  the 
reaction  has  apparently  been  found  to  be  absent.  Such  cases 
should  always  be  investigated,  from  the  point  of  view  of  their 
possibly  being  paratyphoid  fever. 

It  has  been  found  that  the  reaction  is  not  only  obtained  with 
living  bacilli,  but  in  certain  circumstances  also  with  bacilli 
that  have  been  killed  by  heating  at  60°  C.  for  an  hour, — if  a 
higher  temperature  be  used,  sensitiveness  to  agglutination  is 
impaired.  Dreyer  has  introduced  a  simple  technique  which 
enables  an  ordinary  practitioner  provided  with  dead  cultures  to 
carry  out  the  test  for  himself.  The  capacity  is  also  still  retained 
if  a  germicide  be  employed.  Here  Widal  recommends  the 
addition  of  one  drop  of  formalin  to  150  drops  of  culture.  The 
reaction,  however,  tends  to  be  less  complete. 

Besides  the  blood  serum,  it  has  been  found  that  the  reaction 
is  given  in  cases  of  typhoid  fever  by  pericardial  and  pleural 
effusions,  by  the  bile  and  by  the  milk,  and  also  to  a  slight 
degree  by  the  urine.  The  blood  of  a  foetus  may  have  little 
agglutinating  effect,  though  that  of  its  mother  may  have  given 
a  well-marked  reaction ;  sometimes,  however,  the  foetal  blood 
gives  a  well-marked  reaction.  It  may  here  also  be  mentioned 
that  a  serum  will  stand  exposure  for  an  hour  at  58°  C.  without 
having  its  agglutinating  }>ower  much  diminished.  Higher 
temperatures,  however,  cause  the  proj>erty  to  be  lost. 

The  Agglutination  of  Organisms  other  than  the  B.  TyphoxuK 
ly  Typhoid  Serum. — It  was  at  first  thought  that  the  reaction  in 
typhoid  fever  would  afford  a  reliable  method  of  distinguishing 
the  typhoid  bacillus  from  the  b.  coli.  Though  many  races  of 
the  latter  give  no  reaction  with  a  typhoid  serum,  there  are  others 
which  react  positively.  Usually,  however,  a  lower  dilution  and 
a  longer  time  are  required  for  a  result  to  be  obtained,  and  the 
reaction  is  often  incomplete.  It  has  also  been  found  that  other 
organisms  belonging  to  the  typhoid  group  (v.  p.  382)  react  in  a 
similar  way.  The  reaction  as  a  method  of  distinguishing  between 
these  forms  is  thus  not  absolutely  reliable,  but  in  certain  cases 
it  is  of  great  value  in  giving  confirmation  to  other  tests.  The 
important  point  here  is  the  determination  of  the  highest  dilution 
with  which  clumping  is  obtained  (for  methods,  see  p.  120). 
There  is  a  point  in  this  connection  regarding  which  further 


374  TYPHOID  FEVER 

light  is  required.  Many  races  of  b.  coli  in  use  have  been 
isolated  from  typhoid  cases,  and  we  as  yet  do  not  know  what 
effect  this  circumstance  may  have  on  its  subsequent  sensitive- 
ness to  agglutination  by  typhoid  serum.  Again,  Christophers 
has  pointed  out  that  a  large  proportion  of  serum  from  normal 
persons  or  from  those  suffering  from  diseases  other  than  typhoid 
will  clump  the  b.  coli  in  dilutions  of  from  1  :  20  to  1  :  200,  and 
no  doubt  many  of  the  reactions  shown  by  typhoid  sera  towards 
b.  coli  are  due  to  the  pre-existence  in  the  individuals  of  an 
agglutinative  property  towards  the  latter  bacillus. 

With  regard  to  the  value  of  the  serum  reaction  there  is  little 
doubt.  In  nearly  95  per  cent,  of  cases  of  typhoid  it  can  be 
obtained  in  such  a  form  that  no  difficulty  is  experienced  if  the 
precautions  detailed  above  are  observed.  The  causes  of  possible 
error  may  be  summarised  as  follows  :  The  serum  of  the  person 
may  naturally  have  the  capacity  of  clumping  typhoid  bacilli ; 
there  may  have  been  an  attack  of  typhoid  fever  previously  with 
persistence  of  agglutinative  capacity ;  the  case  may  be  one  of 
disease  caused  by  an  allied  bacillus  ;  the  disease  may  have  a 
quite  different  cause,  and  yet  the  serum  may  react  with  typhoid 
bacilli ;  the  disease  may  be  typhoid  fever  and  yet  no  reaction 
may  occur.  The  most  important  of  these  sources  of  error  is  that 
with  which  diseases  caused  by  allied  organisms  are  concerned, 
as  it  is  probable  that  all  the  forms  which  these  take  in  men 
have  not  been  recognised.  The  very  wide  application  of  the 
reaction  has  elicited  the  fact  that  it  is  given  in  many  cases  of 
slight,  transient,  and  ill-defined  febriculte,  which  occur  especially 
when  typhoid  fever  is  prevalent.  Some  of  these  may  be  aborted 
typhoid,  some  may  be  paratyphoid.  There  is  no  doubt  that,  if 
all  the  facts  are  taken  into  account,  the  cases  where  the  reaction 
gives  undoubtedly  correct  information  so  far  outnumber  those  in 
which  an  error  may  be  made  that  it  must  be  looked  on  as  a 
most  valuable  aid  to  diagnosis.  In  conclusion,  here  we  may  say 
that  the  fact  of  a  typhoid  serum  clumping  allied  bacilli  in  no 
way,  so  far  as  our  present  knowledge  goes,  justifies  doubt  being 
cast  on  the  specific  relation  of  the  typhoid  bacillus  to  typhoid 
fever. 

In  connection  with  the  phenomenon  that  a  serum  either  from 
a  normal  person  or  a  typhoid  patient  may  clump  several  varieties 
of  bacteria,  some  points  arise.  The  theoretical  consideration  of 
agglutination  is  reserved  for  the  chapter  on  Immunity,  but  here 
it  -may  be  said  that  agglutinating  properties  may  be  present 
normally  in  a  serum  or  they  may  be  originated  by  an  animal 
baing  infected  with  a  particular  bacterium,  As  the  result  of 


VACCINATION  AGAINST  TYPHOID  375 

injecting  a  bacterium,  not  only  may  agglutinins  capable  of  acting 
on  that  bacterium  appear  in  the  serum,  but  the  serum  may 
become  capable  of  agglutinating  other,  and  especially  kindred, 
bacteria ;  further,  any  normal  agglutinins  for  the  infecting 
bacterium  present  in  the  serum  may  be  increased  in  amount. 
The  agglutinin  acting  on  the  infecting  organism  has  been  called 
the  primary  or  homologous  agglutinin,  while  the  others  have 
been  called  the  secondary  or  heterologous  agglutinins.  But 
besides  what  we  know  to  be  a  fact,  that  infection  by  a  single 
bacillary  species  can  originate  agglutinins  acting  both  on  itself 
and  on  allied  species,  we  must  consider  the  possibility  of 
infections  by  more  than  one  species  occurring  in  an  animal,  e.g. 
b.  typhosus  with  b.  coli  or  with  b.  paratyphosus  (vide  infra).  In 
such  a  case  each  organism  may  originate  its  primary  agglutinin, 
so  that  the  presence  of  multiple  agglutinins  in  a  serum  may 
really  be  an  indication  of  a  mixed  infection.  Some  attention 
has  been  directed  to  the  diagnosis  and  differentiation  of  these 
conditions.  Castellani  introduced  the  absorption  method  for 
their  investigation  (for  method,  see  p.  121),  and  by  this  means 
studied  the  primary  and  secondary  agglutinins  produced  in 
infections  in  rabbits  ;  he  found  that  when  an  animal  had  been 
infected  with  b.  typhosus  this  organism  would  absorb  from  its 
serum  not  only  the  primary  typhoid  agglutinins  but  also  such 
secondary  agglutinins  as  those  acting  on  the  b.  coli.  If,  how- 
ever, an  animal  had  undergone  infection  with,  say,  both  the 
b.  typhosus  and  the  b.  coli,  then  the  b.  typhosus  could  not  absorb 
from  its  serum  the  b.  coli  (primary)  agglutiuin.  Castellani  thus 
put  forward  the  view  that  by  this  means  primary  could  be 
differentiated  from  secondary  agglutinins,  and  therefore  pure 
could  be  differentiated  from  mixed  infections.  There  is  little 
doubt  that  this  view  possesses  considerable  validity,  though  it  is 
probably  not  of  universal  applicability.  Safe  deductions  can 
only  be  drawn  when  any  serum  is  tested  with  several  species  of 
fairly  closely  related  organisms,  such  as  those  of  the  coli  group. 
Especially  is  it  necessary  that  the  highest  dilutions  in  which 
agglutination  occurs  should  be  compared.  If  such  precautions 
be  adopted,  the  absorption  method  can  be  utilised  for  the  differ- 
entiation of  the  typhoid  and  paratyphoid  organisms  and  their 
infections,  and  for  similar  investigations. 

Vaccination  against  Typhoid. — The  principles  of  the  im- 
munisation of  animals  against  typhoid  bacilli  have  been  applied 
by  Wright  and  Semple  to  man  in  the  following  way : — Typhoid 
bacilli  are  obtained  of  such  virulence  that  a  quarter  of  a  twenty- 
four  hours'  old  sloped  agar  culture  when  administered  hypo- 


376  TYPHOID  FEVER 

dermically  will  kill  a  guinea-pig  of  from  350  to  400  grammes. 
Vaccination  can  be  accomplished  by  such  a  culture  emulsified  in 
bouillon,  and  killed  by  heating  for  five  minutes  at  60°  C.  For 
use,  from  one-twentieth  to  one-fourth  of  the  dead  culture  is 
injected  hypodermically,  usually  in  the  flank.  The  vaccine  now 
used,  however,  actually  consists  of  a  portion  of  a  bouillon  culture 
similarly  treated  (see  p.  133).  The  effects  of  the  injection  are 
some  tenderness  locally  and  in  the  adjacent  lymphatic  glands, 
and  it  may  be  local  swelling,  all  of  which  come  on  in  a  few 
hours,  and  may  be  accompanied  by  a  general  feeling  of  restless- 
ness and  a  rise  of  temperature,  but  the  illness  is  over  in 
twenty-four  hours.  During  the  next  ten  days  the  blood  of  the 
individual  begins  to  manifest,  when  tested,  an  agglutination 
reaction,  and  further,  Wright  has  found  that  usually  after  the 
injection  there  is  a  marked  increase  in  the  capacity  of  the  blood 
serum  to  kill  the  typhoid  bacillus  in  vitro.  These  observations, 
there  is  little  doubt,  indicate  that  the  vaccinated  person  possesses 
a  degree  of  immunity  against  the  bacillus,  a  conclusion  borne 
out  by  the  results  obtained  in  the  use  of  the  vaccine  as  a 
prophylactic  against  typhoid  fever.  Extensive  observations 
have  been  made  in  the  British  army  in  India,  and  in  the  South 
African  War  the  efficacy  of  the  treatment  was  put  to  test. 
Though  in  isolated  cases  not  much  difference  was  observed 
among  those  treated  as  compared  with  those  untreated,  yet  the 
broad  general  result  may  be  said  to  leave  little  doubt  that  on  the 
one  hand  protective  inoculation  diminishes  the  tendency  for 
the  individual  to  contract  typhoid  fever,  and,  on  the  other,  if  the 
disease  be  contracted,  the  likelihood  of  its  having  a  fatal  result 
is  diminished.  Thus,  in  India,  of  4502  soldiers  inoculated,  '98 
per  cent,  contracted  typhoid,  while  of  25,851  soldiers  in  the 
same  stations  who  were  not  inoculated,  2 '54  per  cent,  took  the 
disease.  In  Ladysmith  during  the  siege  there  were  1705 
soldiers  inoculated,  among  whom  2  per  cent,  of  cases  occurred, 
and  10,529  uninoculated,  among  whom  14  per  cent,  suffered 
from  typhoid.  Wright  has  collected  statistics  dealing  in  all 
with  49,600  individuals,  of  whom  8600  were  inoculated,  and 
showed  a  case  incidence  of  2 '25  per  cent.,  with  a  case  mortality 
of  12  per  cent.  ;  in  the  remaining  41,000  uninoculated  the  case 
incidence  was  5 '75  per  cent,  and  the  case  mortality  21  per  cent. 
The  best  results  seemed  to  be  obtained  when  ten  days  after 
the  first  inoculation  a  second  similar  inoculation  was  practised. 
Weight  has  found  that  in  certain  cases  immediately  after 
inoculation  there  is  a  fall  in  the  bactericidal  power  of  the  blood 
(negative  phase),  and  he  is  of  opinion  that  this  indicates  a 


METHODS  OF  EXAMINATION  377 

temporary  increased  susceptibility  to  the  disease.  He  therefore 
recommends  that  when  possible  the  vaccination  should  be  carried 
out  some  time  previous  to  the  exposure  to  infection.  There  can 
be  little  doubt  that  in  this  method  an  important  prophylactic 
measure  has  been  discovered. 

Vaccine  Treatment  of  Typhoid  Fever. — As  in  the  case  of 
other  acute  infections,  vaccines  have  been  recently  used  in  the 
treatment  of  typhoid  fever  during  the  acute  stage  (Leishman 
and  Smallman).  The  method  is  to  inject  hypodermically  100 
million  dead  typhoid  bacilli,  i.e.  a  fifth  of  the  first  dose  used  for 
the  protective  inoculation.  If  the  temperature  shows  a  tendency 
to  fall,  this  may  be  repeated  about  every  four  days.  The  results 
obtained  are  hopeful,  and  justify  the  method  being  further 
applied. 

Antityphoid  Serum. — Chanteim-sse  has  immunised  animals  with  dead 
cultures  of  the  typhoid  bacillus,  and,  having  found  that  their  sera  had 
protective  and  curative  effects  in  other  animals,  lias  used  such  sera 
in  human  cases  of  typhoid  with  apparent  good  result.  In  the  hands 
of  others,  however,  such  a  line  of  treatment  has  not  been  equally 
successful. 

Methods  of  Examination. — The  methods  of  miscroscopic 
examination,  and  of  isolation  of  typhoid  bacilli  from  the  spleen 
]*>st  mortem,  have  already  been  described.  They  may  be  isolated 
from  the  Peyer's  patches,  lymphatic  glands,  etc.,  by  a  similar 
method. 

During  life,  typhoid  bacilli  may  be  obtained  in  culture  in  the 
following  ways : — 

(a)  From  the  Blood. — The  typhoid  bacillus  can  often  be 
isolated  from  the  blood,  especially  during  the  first  week,  by 
ordinary  methods  (see  p.  72).  A  special  method  has  also  been 
used  with  success.  In  this  5  c.c.  of  blood  are  placed  in  10  c.c. 
of  sterilised  ox  bile.  The  mixture  is  incubated  for  from  twenty- 
four  hours  to  a  week,  and  from  time  to  time  the  presence  of  the 
bacillus  is  tested  for  by  sub-culturing  on  such  media  as  those  of 
Conradi  or  MacConkey. 

(l»)  From  the,  Spleen. — This  is  the  most  certain  method  of 
obtaining  the  typhoid  bacillus  during  the  continuance  of  a  case. 
The  skin  over  the  spleen  is  purified,  and,  a  sterile  hypodermic 
syringe  being  plunged  into  the  organ,  there  is  withdrawn  from 
the  splenic  pulp  a  droplet  of  fluid,  from  which  plates  are  made. 
In  a  large  proportion  of  cases  of  typhoid  the  bacillus  may  be 
thus  obtained,  failure  only  occurring  when  the  needle  does  not 
hap]K'n  to  touch  a  bacillus.  Numerous  observations  have  shown 


378  TYPHOID  FEVER 

that,  provided  the  needle  be  not  too  large,  the  procedure  is  quite 
safe.     Its  use,  however,  is  scarcely  called  for. 

(c)  From   the    Urine. — Typhoid    bacilli    are    present    in    the 
urine  in  at   least  25  per  cent,  of  cases,  especially  late  in  the 
disease,  probably  chiefly  when  there  are  groups  in  the  kidney 
substance.     For   methods    of    examining   suspected    urine,    see 
p.  74. 

(d)  From  the  Stools. — During  the  first  ten  days  of  a  case  of 
typhoid  fever,  the  bacilli  can  be  isolated  from  the  stools  by  the 
ordinary  plate  methods — preferably  in  McConkey's  lactose  bile- 
salt  neutral-red  agar,  or  in  the  other  media  described  on  pp. 
47-53.     After  that  period,  though  the  continued  infectiveness  of 
the  disease  indicates  that  they  are  still  present,  their  isolation  is 
difficult.     We  have  seen  that  after  ulceration  is  fairly  estab- 
lished by  the    sloughing   of   the    necrosed  tissue,  the  numbers 
present  in  the  patches  are  much  diminished,  and  therefore  there 
are  fewer  cast  off  into  the  intestinal  lumen,  and  that  in  addition 
there  is  a  correspondingly  great  increase  of  the  b.   coli,  which 
thus  causes  any  typhoid  bacilli  in  a  plate  to  be  quite  outgrown. 
From  the  fact  that  the  ulcers  in  a  case  of  typhoid  may  be  very 
few  in  number,  it  is  evident  that  there  may  be  at  no  time  very 
many  typhoid  bacilli  in  the  intestine.     The  microscopic  examina- 
tion of  the  stools  is  of  course  useless  as  a  means  of  diagnosing 
the  presence  of  the  typhoid  bacillus. 

.Isolation  from  Water  Supplies. — A  great  deal  of  work  has  been  done 
on  this  subject.  It  is  evident  that  if  it  is  difficult  to  isolate  the  bacilli 
from  the  stools,  it  must  a  fortiori  be  much  more  difficult  to  do  so  when 
the  latter  are  enormously  diluted  by  water.  The  b.  typhosus  has,  how- 
ever, been  isolated  from  water  during  epidemics.  The  b.  coli  is,  as 
might  be  expected,  the  organism  most  commonly  present  in  such 
circumstances.  In  the  case  of  both  bacteria,  the  whole  series  of  culture 
reactions  must  be  gone  through  before  any  particular  organism  isolated 
is  identified  as  the  one  or  the  other  ;  probably  there  are  saprophytes 
existing  in  nature  which  only  differ  from  them  in  one  or  two  reactions. 
In  examining  waters,  the  ordinary  plate  methods  are  generally  used,  but 
the  McConkey  or  similar  media  may  be  employed  with  advantage. 
Klein  filters  a  large  quantity  through  a  Berkefeld  filter,  and,  brushing 
off  the  bacteria  retained  on  the  porcelain,  makes  cultures.  A  much 
greater  concentration  of  the  bacteria  is  thus  obtained.  From  time  to  time 
various  substances  have  been  used  with  the  object  of  inhibiting  the  growth 
of  the  b.  coli  without  interfering  with  that  of  the  b.  typhosus.  Most  of 
these  have  not  stood  the  text  of  experience.  Lately  caffeine  has  been 
used  for  this  end.  For  use  in  examining  waters  the  following  is  the 
method  employed  :  To  900  c.c.  of  the  suspected  water  there  are  added 
10  grammes  nutrose  dissolved  in  80  c.c.  of  sterile  water,  and  5  grammes 
of  caffeine  dissolved  in  sterile  distilled  water,  heated  to  80°  C.  and  cooled 
to  55°  C.  before  addition.  After  mixing  the  ingredients  there  is  added 
10  c.c.  of  '1  per  cent,  crystal  violet.  The  flask  is  incubated  at  37°  C.  for 


FOOD-POISONING  BACILLI  379 

twelve  hours,  and  then  plates  of  Conradi-Drigalski  medium  are  inoculated 
from  it.  For  investigation  of  faeces,  a  medium  made  up  as  above  but  with 
ordinary  st<-rilr  water  may  be  inoculated  and  a  similar  procedure  followed. 
On  the  whole  there  is  little  to  be  gained  from  this  attempt  to  isolate  the 
typhoid  bacillus  from  water  in  any  particular  case,  and  it  is  much  more 
useful  for  the  bacteriologist  to  bend  his  energies  towards  the  obtaining 
of  the  indirect  evidence  of  contamination  of  water  by  sewage,  to  the 
nature  of  which  attention  has  been  called  in  Chapter  V. 


THE  PARATYPHOID  AND  FOOD-POISONING  BACILLI. 

In  the  b.  coli  we  have  an  organism  having  a  definite  habitat 
in  the  animal  intestine,  and  presenting  certain  cultural  characters 
by  which  it  may  be  recognised.  We  may  look  on  the  bacillus 
typhosus  as  an  organism  of  the  same  class  whose  cultural 
reactions  as  compared  with  b.  coli  present  somewhat  negative 
characters,  but  which  acquires  definiteness  from  its  association 
with  a  well-known  clinical  condition.  We  have  now  to  deal 
with  a  group  of  organisms  which  occupy  rather  an  intermediate 
position  between  the  two  organisms  referred  to,  and  whose 
cultural  characters  are  such  as  to  make  their  differentiation  from 
either  fairly  practicable.  The  members  of  this  group  have  been 
originally  described  in  association  with  a  variety  of  clinical 
conditions,  but,  notwithstanding,  they  resemble  each  other  so 
closely  that  great  difficulty  arises,  and  the  recognition  of  different 
types  which  in  literature  receive  different  names  can  only  be 
effected  by  the  application  of  the  finest  bacteriological  tests. 
Although  in  cultures  the  different  types  present  slight  differences, 
these  are  not  sufficient  for  the  assignment  of  a  name  to  an 
organism  of  the  class  isolated  from  some  fresh  source,  and,  as  a 
matter  of  fact,  in  modern  work  relating  to  them,  it  is  generally 
impossible  in  identifying  an  organism  to  rely  on  merely  noting 
a  correspondence  with  a  described  type.  The  method  usually 
adopted  is  to  obtain  from  other  workers  cultures  of  what  may 
be  called  the  historic  strains  isolated,  and  by  comparing  the 
organism  under  investigation  with  these,  to  attempt  to  place  it 
in  its  proper  position. 

Organisms  of  the  group  are  of  great  importance,  not  only 
from  their  producing  ordinary  infective  disease  in  man,  but 
because  they  are  the  agents  at  work  in  the  great  majority  of 
the  not  infrequently  occurring  cases  of  illness  usually  described 
as  "  food  poisoning."  l  Such  poisoning  is  often  referred  to  as 
"  ptomaine  poisoning,"  from  the  idea  originally  prevailing  that 

1  A  special  type  of  food  poisoning  is  associated  with  the  Bfn-illi's  l>«tulinm, 
fj.v. 


380  TYPHOID  FEVER 

the  symptoms  were  caused  by  alkaloidal  substances  produced 
during  putrefactive  processes  occurring  in  meat.  Certain  cases 
of  illness  arising  within  an  hour  or  two  of  the  taking  of  tainted 
meat  may  be  due  to  poisons  of  such  a  kind,  but  in  the  great 
majority  of  single  or  multiple  cases  of  illness  traceable  to  food, 
the  symptoms  do  not  appear  so  rapidly,  and  are  associated  with 
the  multiplication  in  the  intestine  of  organisms  of  the  type  now 
under  consideration,  and  it  may  be  also  with  an  infection  of  the 
blood.  In  such  cases,  the  meat  at  fault  may  not,  to  taste  or 
smell,  present  any  unusual  features,  but  very  often  there  can  be 
isolated  from  it  an  organism  identical  with  organisms  derived 
from  the  sick  individuals.  Sometimes  it  has  been  proved  that 
the  animals  from  which  the  meat  was  derived  have  been  suffer- 
ing from  illnesses  probably  due  to  the  organisms  subsequently 
found,  but  this  has  not  always  been  the  case,  healthy  meat  being 
here  contaminated  by  contact  with  infective  matter.  The  foods 
giving  rise  to  poisoning  usually  belong  to  the  preserved  food 
class,  or  consist  of  sausages  or  similar  products.  There  is  every 
reason  to  believe  that  the  organisms  in  question  may  not  be 
killed  in  the  ordinary  processes  of  cooking,  in  which  the  internal 
parts  of  the  meat  may  not  reach  the  temperature  of  blood 
coagulation. 

The  organisms  included  in  the  paratyphoid  and  food-poisoning 
group  are  as  follows  :  The  paratyphoid  bacillus,  varieties  A  and 
B,  originally  isolated  from  pathological  conditions  in  man ; 
bacillus  enteriditis  Gaertner,  isolated  from  meat-poisoning  cases  ; 
bacillus  ^Ertryck,  also  isolated  from  meat  poisoning;  bacillus 
suipestifer  (Salmon's  bacillus  of  hog  cholera) ;  psittacosis  bacillus, 
occurring  in  a  disease  of  parrots ;  bacillus  typhi  murium,  isolated 
by  Loffler  from  an  epidemic  of  enteritis  in  mice ;  and  Danysz's 
bacillus,  isolated  from  an  epidemic  in  field  mice,  and  used  by 
him  for  originating  epidemics  in  rats.  The  pathological  effects 
produced  by  these  organisms  include,  on  the  one  hand,  general 
septicsemic  manifestations,  and,  on  the  other,  gastro-enteritis. 
The  chief  members  of  the  group  will  be  described  below. 

The  Characters  of  the  Paratyphoid  and  Food-Poisoning 
Bacilli. — These  bacilli  are  all  miscroscopically  indistinguish- 
able from  the  bacillus  typhosus.  They  are  Gram-negative, 
motile  bacilli,  the  flagella  being  sometimes  few  in  number,  and 
they  do  not  form  spores.  On  ordinary  media,  growths  have  the 
general  character  of  those  of  the  b.  coli  and  b.  typhosus,  some 
members  in  certain  reactions  resembling  the  one,  and  in  others 
resembling  the  other.  Opinion  differs  as  to  their  capacity  to 
form  indol,  but  usually  the  reaction  to  this  test  is  negative. 


FOOD-POISONING  BACILLI  381 

The  methods  for  the  isolation  of  the  members  of  the  group 
vary  with  the  nature  of  the  infected  material  to'  be  examined. 
In  the  case  of  abscesses  caused  by  the  paratyphoid  bacillus, 
the  organism  is  usually  accidentally  discovered  during  the 
application  of  ordinary  methods.  When  deliberate  search  for  a 
member  of  the  group  is  required,  usually  either  the  faeces  or  the 
blood  constitutes  the  material  to  be  examined.  In  the  former 
case,  advantage  is  taken  of  the  fact  that  the  food-poisoning 
bacilli  do  not  ferment  lactose.  Thus,  if  McConkey  bile-salt 
lactose-agar  plates  (p.  50)  be  used,  the  organisms  sought  for 
will  appear  as  colourless  colonies  which  can  be  picked  off  for 
systematic  investigation.  In  the  case  of  blood,  ordinary  methods 
will  prove  sufficient. 

Capacity  for  fermenting  sugars  has  been  largely  applied  in 
work  on  this  group.  All  the  members  produce  practically  the 
same  reactions.  They  originate  acid  and  gas  in  glucose,  lajvu- 
lose,  sorbite,  mannite,  dextrin,  maltose,  dulcite,  galactose  and 
arabinose,  like  b.  coli,  but  produce  no  change  in  lactose,  cane- 
sugar,  salicin  or  inulin.  Although  differences  in  fermenting 
capacity  have  been  noted  in  different  strains,  the  existence  of 
such  cannot  be  relied  upon  for  differentiating  members  of  the 
group  from  one  another.  The  sugar  reactions  are  only  of  use 
in  demarcating  the  lines  between  the  food-poisoning  group  and 
b.  coli  on  the  one  hand,  and  b.  typhosus  on  the  other.  The 
differentiation  of  members  of  the  group  can  only  be  effected  by 
applying  the  agglutination  tests  to  the  serum  of  animals  suffer- 
ing from  natural  or  artificial  infection.  The  chief  point  here  is 
that  in  such  infections,  the  occurrence  of  group  agglutinins  in 
the  serum  is  much  in  evidence.  Herein  lies  the  necessity  for 
having  at  hand  the  historic  strains  of  the  organisms  referred  to 
above.  In  dealing  with  an  organism,  it  is  first  of  all  advisable 
to  take  the  serum  of  the '  infected  individual,  estimate  the 
highest  dilution  with  which  it  clumps  the  strain  isolated,  and 
compare  the  result  obtained  with  the  effect  of  the  serum  on  the 
historic  strains.  The  unknown  strain  is  most  likely  to  be  allied 
to  that  strain  which  is  agglutinated  by  a  similar  dilution  of  the 
serum  used.  Frequently,  in  the  investigation  of  an  organism, 
it  is  necessary  to  inject  it  into  an  animal  and  study  the 
agglutinating  properties  of  its  serum  on  the  infecting  strain 
and  upon  allied  organisms.  Here  considerable  information  may 
be  obtained  by  the  use  of  the  absorption  method.  If  from  such 
a  serum,  for  instance,  an  unknown  organism  has  absorptive 
qualities  similar  to  that  of  a  historic  Gaertner,  its  being  named 
a  Gaertner  bacillus  would  be  justified.  It  is  customary  in  any 


382  TYPHOID  FEVER 

case  to  note  the  action  of  a  typhoid  serum  on  an  organism  under 
investigation,  and  also  the  action  on  the  typhoid  bacillus  of  an 
antiserum  to  the  unknown  organism. 

The  Paratyphoid  Bacillus. — This  organism,  which  was  when 
first  described  often  called  the  paracolou  bacillus,  was 
primarily  isolated  from  abscesses  occurring  in  apparently  non- 
typhoid  cases.  Widal  noted  its  resemblances  to  b.  typhosus  and 
b.  coli,  from  the  latter  of  which  it  differed  in  not  producing 
indol  and  in  not  fermenting  lactose.  Gywnn  first  isolated  it 
from  the  blood  of  a  case  presenting  typhoid  symptoms,  and  since 
then  it  has  been  recognised  as  being  the  probable  cause  of  the 
disease  effects  in  about  3  per  cent,  of  cases  which  clinically  are 
to  be  described  as  typhoid  fever.  The  case  mortality  in  para- 
typhoid fever  is  low,  being  only  from  1  to  2  per  cent.  The 
organism  has  been  isolated  from  the  blood,  the  roseolar  spots, 
and  from  the  stools.  Several  strains  showing  slight  differences 
in  culture  reactions  have  been  obtained.  Of  these  the  two 
chief  are  "  paratyphoid  A "  and  "  paratyphoid  B,"  the  latter 
being  of  commonest  occurrence ;  these  appear  to  present  slight 
cultural  differences.  On  gelatin,  agar,  and  potato,  A  resembles 
b.  typhosus,  B  resembles  b.  coli ;  in  litmus  milk  A  produces 
slight  permanent  acidity,  while  after  the  third  day,  in  the  case 
of  B,  acidity  gives  place  to  alkalinity ;  on  sugars  the  ferment- 
ative activity  of  B  is  greater  than  that  of  A.  Generally  speak- 
ing, the  characters  of  both  are  those  of  the  group  to  which  they 
belong.  With  regard  to  agglutinating  reactions,  the  serum  of 
a  paratyphoid  patient  will  agglutinate  the  bacillus  in  high 
dilutions.  Observations  on  the  behaviour  of  such  sera  towards 
the  b.  typhosus  have  in  different  cases  yielded  some  discordant 
results,  but  usually  a  very  much  stronger  concentration  is 
necessary  to  give  clumping,  and  often  a  paratyphoid  serum  will 
not  clump  the  typhoid  bacillus  except  in  such  concentrations  as 
might  give  similar  effects  when  normal  sera  are  under  observation. 
When  any  serum  clumps  both  the  paratyphoid  and  the  typhoid 
bacilli,  the  more  closely  the  fmaximal  clumping  dilutions  corre- 
spond, the  more  likely  is  the  case  to  be  typhoid  fever ;  on  the 
other  hand,  if  a  high  dilution  will  clump  the  paratyphoid 
bacillus,  while  a  low  dilution  is  necessary  for  the  typhoid 
bacillus,  then  the  case  is  likely  to  be  paratyphoid  fever.  With 
regard  to  the  effects  of  other  sera  on  the  paratyphoid  bacillus, 
it  may  be  said  that  usually  a  typhoid  serum  will  require  to  be 
used  in  greater  concentration  to  clump  this  bacillus  than  is 
necessary  to  obtain  an  effect  with  the  typhoid  bacillus  itself. 
Similar  effects  are  observed  when  the  sera  of  animals  immunised 


BACILLUS  EXTERITIDIS  383 

against  Gaertner's  bacillus  or  the  bacillus  of  psittacosis  are  used. 
In  all  serum  tests  the  essential  point  is  that  deductions  should 
only  be  based  on  comparative  observations  of  the  highest 
dilutions  in  which  a  clumping  effect  is  produced  with  any  series 
of  organisms  compared. 

While  the  paratyphoid  bacillus  originates  a  disease  resembling 
typhoid  fever,  it  has  also  been  found  in  the  stools  of  typhoid 
patients,  and  mixed  infections  may  thus  occur.  Both  organisms 
have  been  observed  together  in  the  stools  in  typhoid  carriers,  and 
pure  paratyphoid  carriers  are  also  stated  to  occur.  A  meat 
poisoning  epidemic  attributed  to  the  paratyphoid  bacillus  has 
been  reported.  Besides  the  septic  cases  already  alluded  to,  the 
organism  has  been  isolated  from  cases  of  bone  abscess,  from 
orchitis,  and  in  Widal's  case  from  a  thyroid  abscess,  and  in  such 
cases  the  history  of  a  previous  typhoid-like  illness  may  not  be 
elicited.  It  has  also  been  found  in  ordinary  faeces.  In  animal 
experiments  it  produces  in  rabbits  and  guinea-pigs  a  fatal  illness 
of  a  septictemic  type  with  serous  inflammations. 

Bacillus  Enteritidis  (Gaertner). — In  1888,  Gaertner,  in 
investigating  a  number  of  cases  of  gastro-enteritis  resulting  from 
eating  the  flesh  of  a  diseased  cow,  isolated,  from  the  meat  and 
from  the  spleen  of  a  man  who  died,  a  bacillus  closely  resem- 
bling the  typhoid  bacillus.  Since  then,  in  a  great  number  of 
similar  outbreaks,  similar  bacilli  have  been  found  both  in  the 
stools  and  in  the  organs.  The  cultural  characters  are  those  of 
the  group,  except  that  in  some  strains  the  presence  of  an  effect  on 
lactose  has  been  observed.  Here  again  much  information  may 
be  obtained  from  the  agglutinating  properties  of  the  serum. 
It  has  also  been  found  that  the  serum  of  persons  suffering  from 
meat  poisoning  sometimes  clumps  the  typhoid  bacillus,  though 
a  higher  concentration  is  required  than  in  the  case  of  Gaertner's 
bacillus.  The  Gaertner  group  of  organisms  is  very  pathogenic 
for  laboratory  animals.  Often,  whatever  the  channel  of  infec- 
tion, there  is  intense  haemorrhagic  enteritis,  and  very  usually 
there  is  a  septicaemia  with  the  occurrence  of  serous  inflammations ; 
the  bacilli  are  recoverable  from  the  solid  organs  and  often  from 
the  blood.  In  man,  as  the  name  of  the  bacillus  indicates,  the 
symptoms  are  centred  in  the  intestine,  where  there  is  usually 
marked  inflammation  of  the  mucous  membrane,  sometimes 
attended  with  haemorrhage  into  it ;  evidence  of  a  septicaemic 
condition  may  also  exist.  Infection  may  take  place  by  the 
bacillus  itself,  and  here  the  illness  usually  appears  within 
twenty-four  hours  of  the  food  being  partaken  of,  but  symptoms 
may  appear  almost  at  once,  in  which  case  they  are  no  doubt  due 


384  TYPHOID  FEVER 

to  the  action  of  toxins ;  here  it  is  important  to  note  that  the 
poisons  formed  by  this  group  of  organisms  are  relatively  heat- 
resisting,  so  that  boiling  for  a  time  does  not  destroy  the  toxicity. 

The  Psittacosis  Bacillus. —When  parrots  are  imported  from  the 
tropics  in  large  numbers,  many  die  of  a  septicaemio  condition  in  which  an 
enteritis,  it  may  be  hsemorrhagic,  is  a  marked  feature.  There  is  intense 
congestion  of  all  the  organs  and  peritoneal  ecchymoses.  From  the 
spleen,  bone  marrow,  and  blood  there  has  been  isolated  a  bacillus  having 
the  group  characters,  except  that  here  also  an  effect  on  lactose  has 
been  described.  The  parrot  is  most  susceptible  to  its  action,  but  it 
also  causes  a  fatal  h?emorrhagic  septicaemia  in  guinea-pigs,  rabbits,  mice, 
pigeons,  and  fowls,  the  bacilli  after  death  being  chiefly  in  the  solid 
organs.  From  affected  parrots  the  disease  appears  to  be  readily 
communicable  to  man,  chiefly,  it  is  probable,  from  the  feathers  being 
soiled  by  infective  excrement.  Several  small  epidemics  have  been 
recognised  and  investigated  in  Paris.  After  about  ten  days'  incubation, 
headache,  fever,  and  anorexia  occur,  followed  by  great  restlessness, 
delirium,  vomiting,  often  diarrhoea,  and  albuminuria.  Frequently 
broncho-pneumonia  supervenes,  and  a  fatal  result  has  followed  in  about 
a  third  of  the  cases  observed.  The  organism  has  been  isolated  from  the 
blood  of  the  heart.  The  psittacosis  bacillus  is  evidently  one  of  the 
typhoid  group,  a  fact  which  is  further  borne  out  by  the  observation  tlmt 
it  may  be  clumped  by  a  typhoid  serum.  The  clumping  is,  however,  said 
often  to  be  incomplete,  as  the  bacilli  between  the  clumps  may  retain 
their  motility.  It  differs  from  the  typhoid  bacillus  in  its  growth  on 
potatoes  and  in  its  pathogenicity. 

Danysz's  Bacillus  and  Rat  Viruses.  —  Danysz  isolated  from  an 
epizootic  in  field  mice  an  organism  of  this  group,  which  he  introduced 
for  the  purpose  of  killing  rats  by  originating  in  them  through  feeding  a 
similar  epizootic,  and  several  viruses  of  this  kind  are  in  commercial  use 
for  this  purpose.  These  have  been  investigated  by  Bainbridge,  who, 
however,  finds  that  they  owe  any  efficiency  they  possess  to  two  organ- 
isms, the  bacillus  Aertryck  and  the  bacillus  enteriditis  of  Caertner.  The 
efficacy  of  such  agents  varies,  and  the  mortality  in  artificially  originated 
epizootics  is  from  20  to  50  per  cent.  Sometimes,  apparently  under 
natural  conditions,  rats  develop  an  immunity  to  those  viruses,  and  it  is 
doubtful  whether  they  are  entirely  innocuous  to  other  animals  which 
may  partake  of  the  food  containing  them. 

BACILLARY  DYSENTERY. 

Dysentery  has  for  long  been  recognised  as  including  a  number 
of  different  pathological  conditions,  and  within  more  recent  times 
amoebic  and  non-amoebic  forms  have  been  distinguished.  Of  the 
latter,  bacteria  have  been  believed  to  be  the  causal  agents,  and  an 
organism  described  by  Shiga  in  1898  has  almost  certainly  been 
established  as  the  cause  of  a  large  proportion  of  cases.  Shiga's 
observations  were  made  in  Japan,  and  confirmatory  results  have 
been  obtained  by  Kruse  in  Germany,  by  Flexner  and  by  Strong 
and  Harvie  in  the  Philippine  Islands,  and  more  recently  by  Vedder 


BACILLUS  DYSENTERIC  385 

and  Duval  in  the  United  States.  It  is  now  further  recognised 
that  the  epidemics  of  dysentery  which  from  time  to  time  occur 
in  lunatic  asylums  are  usually  due  to  bacilli  of  this  type,  and  in 
America  the  organism  has  been  demonstrated  in  summer 
diarrhoea  in  children.  The  evidence  for  the  relationship  of  the 
organism  to  the  disease  consists  chiefly  in  the  apparently  con- 
stant presence  of  the  organism  in  the  dejecta  in  this  form  of 
dysentery,  and  the  agglutination  of  the  organism  by  the  serum 
of  patients  suffering  from  the  disease,  but  confirmatory  evidence 
has  also  come  from  animal  experimentation.  From  different 
epidemics  a  great  many  different  strains  of  the  dysentery  bacillus 
have  been  obtained,  but  these  all  possess  common  characters  and 
'are  undoubtedly  closely  related  to  one  another.  The  various 
strains  resolve  themselves  into  two  chief  groups,  whose  differences 
lie  in  their  behaviour  towards  certain  sugars,  in  their  capacities 
of  forming  indol,  and  in  their  agglutinating  reactions.  The 
relation  of  amoebae  to  dysentery  will  be  discussed  in  the 
Appendix. 

Bacillus  Dysenteriae.  —  Morphological  Characters.  —  This 
bacillus  morphologically  closely  resembles  the  typhoid  bacillus, 
but  is  on  the  whole  somewhat  plumper,  and  filamentous  forms 
are  comparatively  rare.  Involution  forms  sometimes  occur, 
especially  in  glucose  agar.  Most  observers  have  found  no  trace 
of  motility,  whilst  others  say  that  it  is  slightly  motile.  Vedder 
and  Duval  have,  however,  by  a  modification  of  Van  Ermengen's 
process,  demonstrated  in  the  case  of  one  strain  the  presence  of 
numerous  lateral  flagella,  which  are  of  great  fineness,  but  of 
considerable  length.  No  spore  formation  occurs ;  the  organism 
is  stained  readily  by  the  ordinary  dyes,  but  is  decolorised  by 
Gram's  method. 

Cultural  Characters. — In  these  also  considerable  resemblance 
is  presented  to  the  typhoid  bacillus.  In  gelatin  a  whitish  line  of 
growth  occurs  along  the  puncture,  but  the  superficial  film-like 
growtli  is  usually  absent,  or  at  least  poorly  marked.  In  plate 
cultures  also  the  superficial  growths  are  smaller  and  have  less  of 
the  film-like  character,  than  those  of  the  typhoid  organism.  On 
ayar,  growth  occurs  as  a  smooth  film  with  regular  margins,  but 
after  two  or  three  days,  especially  if  the  surface  be  moist,  Vedder 
and  Duval  describe  an  outgrowth  of  lateral  offshoots  on  the 
surface  of  the  medium.  On  agar  plates  the  colonies  resemble 
those  of  the  typhoid  organism,  being  of  smaller  size  and  less 
'»)iaijue  than  those  of  the  bacillus  coli.  In  peptone  bouillon  a 
uniform  haziness  is  produced.  As  has  l^een  indicated,  different 
strains  of  the  bacillus  behave  differently  towards  different  suyarx, 

25 


386  TYPHOID  FEVER 

and  the  results  of  all  observers  do  not  agree,  so  that  only  general 
statements  can  be  made.  Without  going  into  the  question  of 
the  particular  strains  to  be  placed  in  the  two  groups,  we  may  say 
that,  roughly,  these  may  be  classified  into  the  Shiga-Kruse  group 
and  the  Flexner  group.  All  produce  acid  in  peptone-glucose  and 
in  taurocholate  peptone-glucose ;  none  produce  change  in  lactose 
or  cane-sugar.  The  Shiga  group  do  not  produce  acid  in  maltose  or 
mannite,  while  the  Flexner  group  do,  and,  generally  speaking,  the 
former  do  not  produce  indol,  while  the  latter  do.  Forms  inter- 
mediate between  the  two  groups  occur.  There  is  never  any 
evolution  of  gas  observed  in  sugar  media.  In  litmus  milk  there 
is  developed  at  first  a  slight  degree  of  acidity,  which  is  followed 
by  a  phase  of  increased  alkalinity  ;  no  coagulation  of  the  milk ' 
ever  occurs.  On  potato  the  organism  forms  a  transparent  or 
whitish  layer,  which,  however,  in  the  course  of  a  few  days  assumes 
a  brownish-red  or  dirty  grey  colour,  with  some  discoloration  of 
the  potato  at  the  margin  of  the  growth. 

Relation  to  the  Disease. — The  organism  has  been  found  in 
large  numbers  in  the  dejecta,  especially  in  the  acute  cases,  where 
it  may  be  present  in  almost  pure  culture.  In  the  thirty-six  cases 
examined,  Shiga  obtained  it  in  thirty-four  from  the  dejecta,  and  in 
the  two  others  post  mortem  from  the  intestinal  mucous  membrane. 
The  organism  does  not  appear  to  spread  deeply  or  to  invade 
the  general  circulation.  In  the  more  chronic  cases  it  is  difficult 
to  obtain,  on  account  of  the  large  number  of  the  bacillus  coli  and 
other  bacteria  present.  Vedder  and  Duval  found  agar  plates  to 
be  the  best  method  of  culture,  these  being  incubated  at  the 
blood  temperature.  They  also  found  that  if  the  colonies  which 
appeared  at  twelve  hours  were  marked  with  a  pencil,  there  was 
a  greater  probability  of  obtaining  the  bacillus  of  dysentery  from 
those  which  appeared  later,  most  of  those  appearing  early  being 
colonies  of  the  bacillus  coli.  McConkey's  agar  medium  with 
lactose  added  may  be  used  for  isolation  from  stools.  A  little  of 
the  fa3ces  is  rubbed  up  in  broth  and  some  of  the  mixture  stroked 
on  the  medium.  The  formation  of  acid  by  the  b.  coli  colonies 
enables  them  to  be  excluded,  and  therefore,  as  the  b.  dysenteriye 
is  not  a  lactose  fermenter,  the  colourless  colonies  which  develop 
after  twenty -four  hours  are  picked  out  for  further  investigation. 

As  already  stated,  both  acute  arid  chronic  cases  are  marked 
by  the  presence  of  this  organism.  In  the  former,  where  death 
may  occur  in  from  one  to  six  days,  the  chief  changes,  according 
to  Flexner,  are  a  marked  swelling  and  corrugation  of  the  mucous 
membrane,  with  haemorrhage  and  pseudo-membrane  at  places. 
There  is  extensive  coagulation-necrosis  with  fibrinous  exuda- 


BACILLUS  DYSENTERIC  387 

tion  and  abundance  of  polymorpho-nuclear  leucocytes,  and  the 
structure  of  the  mucous  membrane,  as  well  as  that  of  the 
muscularis  mucosae,  is  often  lost  in  the  exudation.  There  is 
also  great  thickening  of  the  sub-mucosa,  with  great  infiltration  of 
leucocytes,  these  being  chiefly  of  the  character  of  "  plasma  cells." 
In  the  more  chronic  forms  the  changes  correspond,  but  are 
more  of  a  proliferative  character.  The  mucous  membrane  is 
granular,  and  superficial  areas  are  devoid  of  epithelium,  whilst 
ulceration  and  pseudo-membrane  are  present  in  varying  degree. 
A  feature  of  bacillary  dysentery  is  the  fact  that  abscess  of  the 
liver  does  not  occur  as  a  complication. 

Agglutination. — All  the  above-mentioned  observers  agree  re- 
garding the  agglutination  of  this  bacillus  by  the  serum — that  is, 
in  the  cases  of  dysentery  from  which  the  organism  can  be  cul- 
tivated. The  reaction  may  appear  on  the  second  day,  and  is 
most  marked  after  from  six  to  seven  days  in  the  acute  cases ;  it 
is  usually  given  in  a  dilution  of  from  one  in  twenty  to  one  in 
fifty  within  an  hour,  though  sometimes  much  higher  dilutions 
give  a  positive  result.  In  the  more  chronic  cases  the  reaction 
is  less  marked,  and  here  the  sedimentation  method  is  to  be 
preferred.  It  is  difficult  to  make  any  general  statements  with 
regard  to  the  effects  of  dysenteric  sera  on  the  different  strains 
of  the  bacilli,  but  it  may  be  said  that  generally  a  serum 
agglutinates  the  strain  which  produced  it  and  the  other  strains 
of  the  same  group  in  higher  dilutions  than  it  does  the  strains  of 
the  other  group.  Many  observers  have  found  that  the  serum 
from  a  case  associated  with  strains  of  the  Shiga-Kruse  group 
has  not  agglutinated  strains  of  the  Flexner  group,  and 
corresponding  observations  have  been  made  in  cases  associated 
with  the  Flexner  group.  Often  the  sera  of  animals  immunised 
with  bacilli  have  been  used  for  such  tests,  but  apparently  great 
care  must  be  exercised  in  basing  diagnoses  on  such  observations, 
as  the  sera  vary  in  different  instances  as  regards  their  action  on 
strains  allied  to  that  used  for  injection.  Agglutination  of  the 
organism  has  not  been  obtained  with  serum  from  cases  other 
than  those  of  dysentery,  nor  has  a  similar  bacillus  been  cultivated 
from  such  sources.  The  reaction  is  also  absent  in  those  cases 
of  dysentery  which  are  manifestly  of  amoebic  nature. 

Pathogenic  Properties. — The  organism  is  pathogenic  in  guinea- 
pigs  and  other  laboratory  animals,  but,  in  these,  characteristic 
changes  in  the  intestine  are  often  awanting.  Shiga,  however, 
obtained  such  effects  by  introducing  the  organism  into  the 
stomach  of  young  cats  and  dogs,  and  confirmatory  results  were 
obtained  by  Flexner.  Such  attempts  have  been  specially 


388  TYPHOID  FEVER 

successful  when  the  virulence  of  the  organism  has  been 
previously  exalted  by  intraperitoneal  passage.  In  two  cases, 
apparently  well  authenticated,  a  dysenteric  condition  has 
followed  in  the  human  subject  from  ingestion  of  pure  cultures 
of  the  organism. 

It  is  probable  that  in  the  action  of  the  bacillus  a  toxin  is 
concerned.  If  the  organism  be  grown  for  two  or  three  weeks  in 
an  alkaline  bouillon,  there  appears,  probably  by  autolysis  of  the 
bacteria,  a  toxin  in  the  culture  medium  separable  by  nitration  in 
the  ordinary  way.  The  optimum  alkalinity  is  achieved  by 
adding  '3  per  cent,  of  soda  to  bouillon  neutral  to  litmus,  the 
resulting  precipitate  not  being  removed  ;  free  access  of  oxygen 
is  permitted  during  growth.  Apparently,  the  Shiga-Kruse 
strains  yield  the  most  toxic  nitrates,  and  with  the  Flexner 
strain,  the  results  of  most  observers  show  that  soluble  toxins 
cannot  be  obtained.  The  poison  is  very  toxic  to  animals, 
especially  rabbits,  and  however  introduced  into  the  body  it 
causes  after  an  incubation  period  haemorrhagic  enteritis  with  a 
diphtheritic-like  exudate  on  the  surface  of  the  mucous  membrane. 
Toxins  isolated  from  different  strains  differ  as  regards  the 
animals  for  which  they  are  most  toxic.  The  toxin  is  fairly 
resistant  to  heat,  standing  temperatures  up  to  70°  C.  without 
being  injured. 

It  may  be  said  that  an  aggressive  reaction  (vide  p.  189)  has 
also  been  described  in  the  case  of  the  dysentery  bacillus. 

Immunisation  Experiments. — Both  large  and  small  animals 
have  been  immunised  against  the  bacillus  and  also  against  its 
toxic  nitrates.  In  the  former  case  the  immunisation  has  been 
commenced  either  with  non-lethal  doses  of  living  cultures,  or  with 
cultures  killed  by  heat.  The  nature  of  the  immunisation  is 
probably  complex.  When  cultures  have  been  used,  a  bactericidal 
serum,  in  which  immune  bodies  and  complements  (vide  Immunity) 
are  concerned,  is  developed.  When  the  toxin  is  used  for 
immunisation,  a  serum  protecting  against  the  toxin  is  produced. 
According  to  some  results,  animals  immunised  with  cultures  are 
immune  against  the  toxin,  and  vice  versa.  All  races  of  animals 
do  not  lend  themselves  to  immunisation. 

Considerable  work  has  been  done  in  immunising  large 
animals  (horses,  goats)  against  the  soluble  toxins  of  the 
dysentery  bacillus  with  a  view  to  obtaining  therapeutic  sera. 
Doerr,  using  his  toxin  from  the  Shiga-Kruse  strain,  produced 
in  horses  an  antitoxic  serum  having  protective  and  curative 
properties  in  animals.  This  serum  has  been  used  in  a  number 
of  cases  of  bacillary  dysentery  in  man  with  good  results.  Shiga 


BACILLUS  ENTERITIDIS  SPOROGENES        389 

produced  a  polyvalent  serum  by  injecting  horses  with  agar 
cultures  of  different  strains,  and  states  that  it  has  been  used 
in  Japan  with  good  results.  Further  observation  is  necessary 
as  to  the  therapeutic  effects  in  cases  associated  with  the  Flexner 
strain  of  an  antitoxin  produced  by  the  Shiga  strain. 

It  will  be  seen  that  the  evidence  furnished  is  practically 
conclusive  as  to  the  causal  relationship  between  this  bacillus  and 
one  form  of  dysentery,  a  form,  moreover,  which  is  both  wide- 
spread and  embraces  a  large  proportion  of  cases  of  the  disease ; 
and  especially  of  importance  is  the  fact  that  observations  made 
independently  in  different  countries  have  yielded  practically 
identical  results  on  this  point. 

Bacillus  Dysenteriae  (Ogata). — Ogata  obtained  this  bacillus  in  an 
extensive  epidemic  in  Japan  in  which  no  amoebae  were  present.  He 
found  in  sections  of  the  affected  tissues  enormous  numbers  of  small 
bacilli  of  about  the  same  thickness  as  the  tubercle  bacillus,  but  very  much 
shorter.  These  bacilli  were  sometimes  found  in  a  practically  pure 
condition.  They  were  actively  motile,  and  could  be  stained  by  Gram's 
method.  He  also  obtained  pure  cultures  from  various  cases  and  tested 
their  pathogenic  effects.  They  grew  well  on  gelatin,  at  the  ordinary 
temperature  producing  liquefaction,  the  growth  somewhat  resembling 
that  of  the  cholera  spirillum.  By  injection  into  cats  and  guinea-pigs,  as 
well  as  by  feeding  them,  this  organism  was  found  to  have  distinct 
pathogenic  effects  ;  these  were  chiefly  confined  to  the  large  intestine, 
haemorrhagic  inflammation  and  ulceration  being  produced.  It  still 
remains  to  be  determined  whether  this  organism  has  a  causal  relationship 
to  one  variety  of  dysentery. 

BACILLUS  ENTEEITIDIS  SPOROGENES. 

This  organism  was  first  isolated  by  Klein  from  the  evacuations  in  an 
outbreak  of  diarrhoea  following  the  ingestion  of  milk  which  contained  the 
microbe,  and  it  was  subsequently  found  by  him  in  certain  cases  of 
infantile  diarrhoea  and  of  summer  diarrhoea,  in  certain  instances  in  milk, 
and  as  a  constant  inhabitant  of  sewage  (see  Chap.  V.).  In  films  made 
from  the  stools  in  diarrhoea  cases  where  it  is  present,  it  can  be  micro- 
scopically recognised  as  a  bacillus  1'6  /*  to  4*8  /A  in  length  and  "8  /*  in 
breadth,  staining  by  ordinary  stains  and  retaining  the  dye  in  Gram's 
method.  It  often  contains  a  spore  near  one  of  the  ends,  or  sometimes 
nearer  the  centre.  It  is  slightly  motile,  and  in  cultures  can  be  shown 
to  possess  a  small  number  of  terminal  flagella.  It  grows  well  under 
anaerobic  conditions  in  ordinary  media,  especially  on  those  containing 
reducing  agents.  On  agar  the  colonies  are  circular,  grey,  and  translucent, 
and  under  a  low  power  are  seen  to  have  a  granular  appearance.  On  this 
in- « Hum  spore  formation  does  not  occur,  but  is  easily  obtained  if  the 
organism  is  grown  on  solidified  blood  serum,  which,  further,  is  liquefied 
(luring  growth.  On  gelatin  plates  liquefaction  commences  after  twenty- 
four  hours  at  20°  C.  It  produces  acid  and  gas  in  bile-salt  glucose  media, 
and  in  peptone-salt  solution  containing  glucose  or  mannite.  Spore 
formation  can  be  seen  to  take  place  in  2  per  cent,  dextrose  gelatin,  but 


390  TYPHOID  FEVEE 

the  degree  seems  to  be  in  inverse  ratio  to  the  amount  of  gas  formation. 
Very  typical  is  the  growth  in  milk,  and  it  is  by  this  medium  that 
isolation  can  be  best  effected.  A  small  quantity  of  the  material 
suspected  to  contain  the  bacillus  is  placed  in  15  to  20  c.c.  of  sterile 
milk,  which  is  then  heated  for  ten  minutes  at  80°  C.  to  destroy  all 
vegetative  bacteria  ;  the  tube  is  cooled,  placed  under  anaerobic  con- 
ditions, and  incubated  at  37°  C.  for  from  twenty-four  to  thirty-six  hours. 
If  the  bacillus  be  present  there  is  abundant  gas  formation,  and  almost 
complete  separation  of  the  curd  from  the  whey  takes  place.  The  former 
adheres  to  the  sides  of  the  tube  in  shreds,  and  large  masses  gather  with 
the  cream  on  the  top  of  the  fluid,  all  being  torn  by  the  gas  evolved.  The 
whey  is  only  slightly  turbid,  and  contains  numerous  bacilli.  The  growth 
lias  an  odour  of  butyric  acid.  If  a  small  quantity  (say  1  c.c.)  of 
the  whey  be  injected  into  a  guinea-pig,  the  animal  becomes  ill  in  a  few 
hours  and  dies  in  twenty-four  hours.  At  the  point  of  inoculation,  the 
skin  and  subcutaneous  tissues,  and  sometimes  even  the  subjacent  muscles, 
are  green  and  gangrenous  and  evil-smelling,  there  is  considerable  oedema, 
and  there  may  also  be  gas  formation.  The  exudation  is  crowded  with 
bacilli,  which,  however,  are  not  generally  distributed  in  any  numbers 
throughout  the  body.  These  pathogenic  properties  of  the  bacillus 
enteritidis  sporogenes  are  important  in  its  recognition,  for  its  culture 
reactions  taken  alone  are  very  similar  to  those  of  the  bacillus  butyricus  of 
Botkin. 

SUMMER  DIARRHOEA. 

As  has  been  already  stated,  the  bacillus  of  dysentery,  the 
b.  coli,  and  the  b.  enteritidis  sporogenes  have  been  found 
associated  with  epidemics  of  this  disease.  This  indicates  that 
the  condition  may  be  originated  by  a  variety  of  organisms,  and 
it  is  further  probable  that  the  clinical  features  in  different 
epidemics  vary.  This  is  to  a  certain  extent  illustrated  by  the 
condition  of  the  stools.  In  Britain  these  are  usually  green, 
watery,  slimy,  and  putrid,  without  blood  or  mucus,  but  in  many 
outbreaks  in  America  blood  and  mucus  are  present.  The 
multiple  origin  of  the  disease  has  been  illustrated  by  the  work  of 
Morgan,  who,  in  a  careful  investigation  of  the  disease  in  Britain, 
has  been  unable  to  find  evidence  of  the  dysentery  bacillus  being 
present.  He  has,  however,  very  frequently  (in  63  per  cent. 
of  the  cases  examined)  found  in  the  stools  and  intestine  a 
bacillus  ("  Morgan's  No.  1  bacillus  ")  which  is  a  motile  Gram- 
negative  organism  producing  acid  and  slight  gas  formation  in 
glucose,  laevulose,  and  galactose,  and  no  change  in  mannite, 
dulcite,  maltose,  dextrin,  cane-sugar,  lactose,  inulin,  amygdalin, 
salicin,  arabinose,  raffinose,  sorbite,  or  erythrite ;  it  further 
causes  indol  formation,  and  in  litmus  milk  slowly  originates  an 
alkaline  reaction.  It  produces  diarrhoea  •  and  death  in  young 
rabbits,  rats,  and  monkeys  when  these  animals  are  fed  on 
cultures.  It  is  thus  possible  that  in  this  bacillus  we  have 


REVIEW  OF  THE  COLT-TYPHOID  BACILLI     391 

still  another  cause  of  the  disease.  Morgan  has  found  that  in 
diarrhoea  cases  the  lactose  fermenters,  so  characteristic  of  normal 
faeces,  are  relatively  less  frequent  and  tend  to  be  replaced  by 
non-fermenters  of  lactose.  His  bacillus  has  been  found  in  a 
certain  proportion  of  normal  children,  but  this  especially  during 
the  epidemic  season  ;  it  has  also  been  found  in  flies. 

GENERAL  REVIEW  OF  THE  COLI-TYPHOID  BACILLI. 

A  general  view  of  the  organisms  belonging  to  the  coli-typhoid 
group  which  we  have  now  considered  indicates  a  close  alliance 
between  the  various  members.  All  are  microscopically  indis- 
tinguishable from  one  another,  and  react  negatively  to  the 
Gram  stain.  The  chief  sub-groups  can  be  differentiated  by 
culture  reactions,  of  which  the  action  on  sugars  is  most  important. 
Here  important  information  is  obtained  by  the  study  of  the 
glucose  and  lactose  reactions.  The  typhoid  sub-group  produces 
acid  on  glucose,  but  has  no  action  on  lactose.  The  dysentery 
sub-group  is  similar,  but  is  chiefly  marked  off  from  the  typhoid 
sub-group  by  its  relative  non-motility,  by  its  tendency  to  form 
alkali  after  a  preliminary  acid  development  on  litmus  milk,  and 
by  the  fact  that  it  does  not  ferment  sorbite.  The  food-poisoning 
sub-group  is  differentiated  from  the  typhoid  sub-group  by 
forming  acid  and  gas  in  glucose  and  from  the  coli-sub-group  by 
its  producing  no  change  on  lactose.  The  positive  features  of  the 
coli  sub-group  are  the  formation  of  acid  and  gas  in  both  glucose 
and  lactose. 

From  work  done  not  only  with  bacteria  isolated  from  patho- 
logical conditions,  but  in  connection  with  the  bacteriology  of 
water,  milk,  and  faeces,  it  has  been  found  that  an  enormous 
number  of  organisms  exist,  having  the  capacity  of  fermenting 
glucose  and  lactose,  but  which,  when  further  investigated,  present 
individual  differences.  Much  has  been  done  in  attempting  to 
differentiate  these  so-called  "  lactose  fermenters "  from  one 
another.  Here  the  work  of  McConkey  may  be  taken  as  con- 
stituting one  of  the  best  attempts  at  such  further  classification, 
and  it  has  the  merit  of  simplifying  a  technique  unduly  compli- 
cated l»y  the  use  of  fermentation  tests  in  a  great  series  of  sugars, 
on  which  the  various  sub-groups  have  all  the  same  effect. 
McConkey  is  of  opinion  that  certain  of  the  tests  applied  to  the 
lactose  fermenters  in  reality  give  little  information.  These  are, 
first,  the  growth  on  litmus  whey,  observation  of  which  only 
corroborates  what  is  observed  with  litmus  milk  ;  second,  observa- 
tion of  fluorescence  on  neutral-red  lactose  media  (on  account  of 


392  TYPHOID  FEVER 

the  inconstancy  of  the  occurrence  of  this  change  in  lactose 
fermenters,  and  from  the  fact  that  many  other  bacteria  also 
produce  it) ;  third,  the  reduction  of  nitrates, — this  appears 
to  be  a  common  property  of  nearly  all  the  members  of  the 
group  ;  fourth,  observation  of  differences  in  the  naked  eye  or 
low  power  appearances  on  gelatin ;  these  are  very  inconstant,  and 
different  colonies  of  the  same  organism  may  show  different 
appearances.  On  the  other  hand,  important  information  may 
be  obtained  by  the  observation  of  the  Voges  and  Proskauer 
reaction  (p.  353).  With  regard  to  sugars,  McConkey  concludes 
that  in  the  differentiation  of  the  lactose  fermenters,  the  only 
sugars  necessary  are  lactose,  saccharose,  dulcite,  adonite,  inulin, 
inosite,  and  mannite.  Using  these,  a  preliminary  classification 
can  be  made  from  the  actions  on  cane-sugar  and  dulcite,  and 
four  groups  are  constituted  :  I.  Organisms  not  affecting  either 
cane-sugar  or  dulcite.  II.  Organisms  having  no  action  on  cane- 
sugar,  but  fermenting  dulcite.  III.  Organisms  fermenting 
both  cane-sugar  and  dulcite.  IV.  Organisms  fermenting  cane- 
sugar  but  having  no  action  on  dulcite.  Of  the  first,  the 
bacillus  acidi  lactici  of  Hiippe  may  be  taken  as  a  type ; 
of  the  second,  the  bacillus  coli  communis  of  Escherich ; 
of  the  third,  bacillus  Friedlander ;  of  the  fourth,  the  bacillus 
lactis  aerogenes  and  the  bacillus  cloacae.  Group  IV.  is  further 
sub-divided  into  sub-group  1,  in  which  there  is  no  lique- 
faction of  gelatin  and  an  absence  of  the  Voges  and  Proskauer 
reaction ;  2,  with  no  liquefaction  of  gelatin,  presence  of  Voges 
and  Proskauer's  reaction  (bacillus  lactis  aerogenes) ;  3,  with 
liquefaction  of  gelatin,  presence  of  Voges  and  Proskauer's 
reaction  (bacillus  cloacae) ;  4,  with  liquefaction  of  gelatin  and 
production  of  a  yellow  pigment.  Taking  the  properties  named 
as  type  characteristics,  the  great  mass  of  lactose  fermenters  can 
be  further  differentiated  by  the  application  of  the  other  sugar 
tests.  It  is  well  to  refer  any  organism  found  as  belonging  to 
one  or  other  of  the  types,  as  in  most  cases  no  name  has  been 
assigned.  Examples  are  constantly  met  with  in  work  on  water 
or  faecal  contents. 

Although  many  of  the  named  varieties  were  originally 
described  in  connection  with  other  bacterial  processes,  all  these 
bacteria  are  of  frequent  occurrence,  especially  in  the  human  and 
animal  intestine.  As  in  the  case  of  the  members  of  the  food- 
poisoning  group,  great  difficulty  has  been  experienced  in 
identifying  the  types  from  mere  description,  and  considerable 
complication  has  arisen  from  the  fact  that  before  the  elaboration 
of  the  modern  differentiation  technique,  different  observers 


REVIEW  OF  THE  COLI-TYPHOID  BACILLI      393 

identified  organisms  as  belonging  to  a  classical  type,  which  have 
now  been  found  not  to  conform  in  properties  with  the  historic 
.strains ;  here  again,  it  is  now  customary  during  classification 
work  to  have  at  hand  such  historic  strains  in  order  that  com- 
parative parallel  observations  may  be  made. 

With  regard  to  the  type  strains,  a  few  words  may  be  added. 
The  original  bacillus  coli  communis  of  Escherich  was  isolated 
from  the  intestine  of  newly-born  infants  in  connection  with  the 
first  appearance  of  bacteria  in  the  alimentary  tract.  About  the 
same  time,  an  organism  now  known  as  the  bacillus  neapolitanus 
was  obtained  by  Emmerich  in  an  outbreak  of  choleraic  disease 
in  Naples,  and  this  organism  was  looked  upon  as  identical  with 
Escherich's  bacillus,  but  it  ferments  saccharose,  on  which 
Escherich's  has  no  effect.  The  bacillus  acidi  lactici  of  Hiippe 
was  stated  by  this  observer  to  be  the  chief  cause  of  the  souring 
of  milk.  It  is  now  known  that  a  large  number  of  organisms  of 
the  same  type,  but  differing  slightly  in  cultural  characters,  are 
concerned  in  this  process,  and,  as  a  matter  of  fact,  McConkey 
found  the  presence  of  the  classical  strain  to  be  relatively  infre- 
quent in  milk.  The  bacillus  lactis  aerogenes  was  originally 
described  by  Escherich,  in  connection  with  his  work  on  the 
bacteriology  of  the  intestine  in  children,  as  an  organism  differing 
from  the  ordinary  milk-souring  bacteria  by  its  producing  gas 
from  milk  in  the  absence  of  air.  Although  it  is  a  free  gas- 
producer,  this  property  is  not  specific  for  it,  and  within  recent 
years  it  has  attracted  attention  chiefly  from  its  apparently  being 
closely  allied  to  the  bacillus  pneumonias  of  Friedlander.  Like 
the  latter,  this  organism  is  stated  when  injected  into  animals  to 
appear  in  a  capsulated  form.  Another  member  of  this  group  is 
bacillus  oxytocus  perniciosus,  which  is  said  originally  to  have 
been  isolated  from  milk.  This  organism,  along  with  the  bacillus 
vesiculosus  and  an  organism  denominated  No.  71,  were  found 
by  McConkey  to  be  of  very  common  occurrence  in  human  and 
animal  faeces. 

In  work  of  the  kind  with  which  we  are  dealing,  two  other 
organisms  are  not  infrequently  observed  which  morphologically 
belong  to  the  coli-typhoid  group,  but  neither  of  which  is  a 
lactose  fermenter.  These  are  the  bacillus  faecalis  alcaligenes, 
and  the  bacillus  coli  anaerogenes.  The  reactions  of  these  will  be 
found  in  the  Table  (p.  394).  The  latter  bacillus  somewhat 
resembles  the  typhoid  bacillus,  but  produces  acid  in  lactose 
and  can  be  distinguished  by  agglutinating  reactions. 

When  any  question  arises  regarding  the  relationships  of  an 
organism  isolated  under  saphrophytic  conditions  and  resembling 


394 


TYPHOID  FEVER 


s^an^itsoaij 
put;  saSoA 

iiiiit             +,+                    +111 

-.opuj 

, 

ii 

$$m$  m     i  <  "  ^  < 

5 

d          &    x    x    &          o    d    d'   ^      .    d    d    d 
4;  ^!    3   9    9    3    °    4   4   ^  '  3  ''*    4  ^    .«    °    ' 

« 

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i             .iiiii,                        i 

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d      .    d    d     !       .      !     d    d    d     (       .    d                d 

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,  .",      d     (     d     ,           d     ,     d    d 

, 

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d      -idd      .      -dodo            -dddd 

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i      i      i      ,      ,      ,      ,      i      i     +     ,      i     +     ,      i      i 

•A,moM 

+    +    +    +i      1     +     1      i++i      i      ,     +     i 

Bacterium. 

IT       1?           ^                §       r 

G^rjS                   '            9*             *           •               '            S               '• 

—  P                           |        8l       |       r*         .    ,    g-                  g        g       .S                   0 

'S     7.  i   8-  ®  ^  ^    s  •?    *   3    &   I   *    V,  tj 

5             o^ffiS8-Mo"             Soas^^ 

1  §/  1,  s  1  's  1  si  1    •^i   =  3§! 

S§S.-S^-e|?d^8.2c§-sg^ 
w^1so§S°--s^'5ci-20w^ 

1  S  1  i  1  1  *  1  1  1  •  1  1  1  ?  1 

^M/WMCqp,^^^^^^^^^ 

i                           -\  A 

REVIEW  OF  THE  COLT-TYPHOID  BACILLI     395 

some  definite  pathogenic  type,  important  information  can  often 
be  obtained  by  studying  its  agglutinating  reactions.  In  such  a 
case  the  effect  of  sera  produced  by  the  pathogenic  type  upon 
the  unknown  organism,  and  of  sera  produced  by  injection 
into  animals  of  the  pathogenic  type  in  question,  ought  to  be 
studied. 


CHAPTER   XVI. 

DIPHTHEKIA. 

THERE  is  no  better  example  of  the  valuable  contributions  of 
bacteriology  to  scientific  medicine  than  that  afforded  in  the  case 
of  diphtheria.  Not  only  has  .research  supplied,  as  in  the  case 
of  tubercle,  a  means  of  distinguishing  true  diphtheria  from 
conditions  which  resemble  it,  but  the  study  of  the  toxins  of  the 
bacillus  has  explained  the  manner  by  which  the  pathological 
changes  and  characteristic  symptoms  of  the  disease  are  brought 
about,  and  has  led  to  the  discovery  of  the  most  efficient  means 
of  treatment,  namely,  the  anti-diphtheritic  serum.  . 

Historical. — The  first  account  of  the  bacillus  now  known  to  be  the 
cause  of  diphtheria  was  given  by  Klebs  in  1883,  who  described  its 
characters  in  the  false  membrane,  *but  made  no  cultivations.  It  was 
first  cultivated  by  Loffler  from  a  number  of  cases  of  diphtheria,  his 
observations  being  published  in  1884,  and  to  him  we  owe  the  first 
account  of  its  characters  in  cultures  and  of  some  of  its  pathogenic  effects 
on  animals.  The  organism  is  for  these  reasons  known  as  the  Klebs- 
Loffler  bacillus,  or  simply  as  Loffler's  bacillus.  By  experimental  in- 
oculation with  the  cultures  obtained,  Loffler  was  able  to  produce  false 
membrane  on  damaged  mucous  surfaces,  but  he  hesitated  to  conclude 
definitely  that  this  organism  was  the  cause  of  the  disease,  for  he  did 
not  find  it  in  all  the  cases  of  diphtheria  examined,  he  was  not  able  to 
produce  paralytic  phenomena  in  animals  by  its  injection,  and,  further, 
he  obtained  the  same  organism  from  the  throat  of  a  healthy  child.  This 
organism  became  the  subject  of  much  inquiry,  but  its  relationship  to 
the  disease  may  be  said  to  have  been  definitely  established  by  the 
brilliant  researches  of  Roux  and  Yersin,  which  showed  that  the  most 
important  features  of  the  disease  could  be  produced  by  means  of  the 
separated  toxins  of  the  organism.  Their  experiments  were  published  in 
1888-90.  Further  light  has  been  thrown  on  the  subject  by  the  work  of 
Sidney  Martin,  who  has  found  that  there  can  be  separated  from  the 
organs  in  cases  of  diphtheria  substances  which  act  as  nerve  poisons,  and 
also  produce  other  phenomena  met  with  in  diphtheria. 

General  Facts. — Without  giving  a  description  of  the  patho- 
logical changes  in  diphtheria,  it  will  be  well  to  mention  the  out- 
standing features  which  ought  to  be  considered  in  connection 

396 


BACILLUS  DIPHTHERIA  397 

with  its  bacteriology.  In  addition  to  the  formation  of  false 
membrane,  which  may  prove  fatal  by  mechanical  effects,  the 
chief  clinical  phenomena  are  the  symptoms  of  general  poisoning, 
great  muscular  weakness,  tendency  to  syncope,  and  albuminuria  ; 
also  the  striking  paralyses  which  occur  later  in  the  disease, 
and  which  may  affect  the  muscles  of  the  pharynx,  larynx,  and 
eye,  or  less  frequently  the  lower  limbs  (being  sometimes  of 
paraplegic  type),  all  these  being  grouped  together  under  the 
term  "  post-diphtheritic  paralyses."  It  may  be  stated  here  that 
all  these  conditions  have  been  experimentally  reproduced  by  the 
action  of  the  bacillus  of  diphtheria,  or  by  its  toxins.  Other 
bacteria  are,  however,  concerned  in  producing  various  secondary 
inflammatory  complications  in  the  region  of  the  throat,  such  as 
ulceration,  gangrenous  change,  and  suppuration,  which  may  be 
accompanied  by  symptoms  of  general  septic  poisoning.  The 
detection  of  the  bacillus  of  Loffler  in  the  false  membrane  or 
secretions  of  the  mouth  is  to  be  regarded  as  supplying  the  only 
certain  means  of  diagnosis  of  diphtheria. 

Bacillus  Diphtherise. — Microscopical  Characters. — If  a  film 
preparation  be  made  from  a  piece  of  diphtheria  membrane  (in 
the  manner  described  below)  and  stained  with  methylene-blue, 
the  bacilli  are  found  to  have  the  following  characters  : — They  are 
slender  rods,  straight  or  slightly  curved,  and  usually  about  3  /n 
in  length,  their  thickness  being  a  little  greater  than  that  of 
the  tubercle  bacillus.  The  size,  however,  varies  somewhat  in 
different  cases,  and  for  this  reason  varieties  have  been  dis- 
tinguished as  small  and  large,  and  even  of  intermediate  size. 
It  is  sufficient  to  mention  here  that  in  some  cases  most  are 
about  3  /A  in  length,  whilst  in  others  they  may  measure  fully 
5  ft.  Corresponding  differences  in  size  are  found  in  cultures. 
They  stain  deeply  with  the  blue,  sometimes  being  uniformly 
coloured,  but  often  showing,  in  their  substance,  little  granules 
more  darkly  stained,  so  that  a  dotted  or  beaded  appearance  is 
presented.  Sometimes  the  ends  are  swollen  and  more  darkly 
stained  than  the  rest;  often,  however,  they  are  rather  tapered 
off  (Fig.  111).  In  some  cases  the  terminal  swelling  is  very 
marked,  so  as  to  amount  to  clubbing,  and  with  some  specimens 
of  methylene-blue  these  swellings  and  granules  stain  of  a  violet 
tint.  Distinct  clubbing,  however,  is  less  frequent  than  in 
cultures.  There  is  a  want  of  uniformity  in  the  appearance  of 
the  bacilli  when  compared  side  by  side.  They  usually  lie 
irregularly  scattered  or  in  clusters,  the  individual  bacilli  being 
disposed  in  all  directions.  Some  may  be  contained  within 
leucocytes.  They  do  not  form  chains,  but  occasionally  forms 


398  DIPHTHERIA 

longer  than  those  mentioned  may  be  found,  and  these  specially 
occur  in  the  spaces  between  the  fibrin  as  seen  in  sections. 

Distribution  of  the  Bacillus. — The  diphtheria  bacillus  may 
be  found  in  the  membrane  wherever  it  is  formed,  and  may  also 
occur  in  the  secretions  of  the  pharynx  and  larynx  in  the  disease. 
It  may  be  mentioned  that  distinctions  formerly  drawn  between 
true  diphtheria  and  non-diphtheritic  conditions  from  the  appear- 


»•. 


FIG.  111.— Film  preparation  from  diphtheria  membrane,  showing 
numerous  diphtheria  bacilli.  One  or  two  degenerated  forms  are  seen 
near  the  centre  of  the  field.  (Cultures  made  from  the  same  piece  of 
membrane  showed  the  organism  to  be  present  in  practically  pure 
condition.) 

Stained  with  methylene-blue.       x  1000. 

ance  and  site  of  the  membrane,  have  no  scientific  value,  the  only 
true  criterion  being  the  presence  of  the  diphtheria  bacillus.  The 
occurrence  of  a  membranous  formation  produced  by  streptococci 
has  already  been  mentioned  (p.  212). 

In  diphtheria  the  membrane  has  a  somewhat  different 
structure,  according  as  it  is  formed  on  the  surface  covered  with 
stratified  squamous  epithelium,  as  in  the  pharynx,  or  on  a  surface 
covered  by  ciliated  epithelium,  as  in  the  trachea.  In  the  former 


DISTRIBUTION  OF  THE  BACILLUS      -     399 

-it  nation  necrosis  of  the  epithelium  occurs  either  uniformly  or 
in  patches,  and  along  with  this  there  is  marked  inflammatory 
reaction  in  the  connective  tissue  beneath,  attended  by  abundant 
iibrinous  exudation.  The  necrosed  epithelium  becomes  raised 
up  by  the  fibrin,  and  its  interstices  are  also  filled  by  it.  The 
fibriuous  exudation  also  occurs  around  the  vessels  in  the  tissue 
beneath,  and  in  this  way  the  membrane  is  firmly  adherent.  In 


Kic.  ll^.--Sfction  through  a  diphtheritic  membrane  in  trachea, 
showing  diphtheria  bacilli  (stained  darkly)  in  clumps,  and  also 
>c 'uttered  amongst  the  fibrin.  Some  streptococci  are  also  shown. 
towards  the  surface  on  the  left  side. 

Stained  by  Gram's  method  and  Bismarck-brown.       x  1000. 

the  trachea,  on  the  other  hand,  the  epithelial  cells  rapidly 
become  shed,  and  the  membrane  is  found  to  consist  almost 
exclusively  of  fibrin  with  leucocytes,  the  former  arranged  in  a 
reticulated  or  somewhat  laminated  manner,  and  varying  in 
density  in  different  parts.  The  membrane  lies  uj)on  the  base- 
ment membrane,  and  is  less  firmly  adherent  than  in  the  case  of 
the  pharynx. 

The  position   of   the   diphtheria  bacilli  varies  somewhat   in 
different  cases,  but  they  are  most  frequently  found  lying  in  oval 


400  DIPHTHERIA 

or  irregular  clumps  in  the  spaces  between  the  fibrin,  towards  the 
superficial,  that  is,  usually,  the  oldest  part  of  the  false  membrane 
(Fig.  112).  There  they  may  be  in  a  practically  pure  condition, 
though  streptococci  and  occasionally  some  other  organisms  may 
be  present  along  with  them.  They  may  occur  also  in  deeper 
parts,  but  are  rarely  found  in  the  fibrin  around  the  blood  vessels. 
On  the  surface  of  the  membrane  they  may  be  also  seen  lying  in 
large  numbers,  but  are  there  accompanied  by  numerous  other 
organisms.  Occasionally  a  few  bacilli  have  been  detected  in  the 
lymphatic .  glands.  As  Loftier  first  described,  they  may  be 
found  after  death  in  pneumonic  patches  in  the  lung,  these 
being  due  to  a  secondary  extension  by  the  air  passages.  They 
have  also  been  occasionally  found  in  the  spleen,  liver,  and 
other  organs  after  death.  This  occurrence  is  probably  to 
be  explained  by  an  entrance  into  the  blood  stream  shortly 
before  death,  similar  to  what  occurs  in  the  case  of  other 
organisms,  e.g.  the  bacillus  coli  communis.  The  diphtheria 
bacillus  may  also  infect  other  mucous  membranes.  It  is  found 
in  true  diphtheria  of  the  conjunctiva,  and  may  also  occur 
in  similar  affections  of  the  vulva  and  vagina ;  some  of  these 
cases  have  been  treated  successfully  with  diphtheria  antitoxin. 
The  pseudo-diphtheria  bacillus,  however,  may  also  occur  in  these 
situations. 

Association  with  other  Organisms. — The  diphtheria  organism  is 
sometimes  present  alone  in  the  membrane,  but  more  frequently 
is  associated  with  some  of  the  pyogenic  organisms,  the  strepto- 
coccus pyogenes  being  the  commonest.  The  staphylococci,  and 
occasionally  the  pneumococcus  or  the  bacillus  coli,  may  be 
present  in  some  cases.  Streptococci  are  often  found  lying  side 
by  side  with  the  diphtheria  bacilli  in  the  membrane,  and  also 
penetrating  more  deeply  into  the  tissues.  In  some  cases  of 
tracheal  diphtheria  we  have  found  streptococci  alone  at  a  lower 
level  in  the  trachea  than  the  diphtheria  bacilli,  where  the 
membrane  was  thinner  and  softer,  the  appearance  in  these  cases 
being  as  if  the  streptococci  acted  as  exciters  of  inflammation  and 
prepared  the  way  for  the  bacilli.  It  is  still  a  matter  of  dispute 
as  to  whether  the  association  of  the  diphtheria  bacillus  with  the 
pyogenic  organisms  is  a  favourable  sign  or  the  contrary,  though 
on  experimental  grounds  the  latter  is  the  more  probable.  We 
know,  however,  that  some  of  the  complications  of  diphtheria 
may  be  due  to  the  action  of  pyogenic  organisms.  The  extensive 
swelling  of  the  tissues  of  the  neck,  sometimes  attended  by 
suppuration  in  the  glands,  and  also  various  hsemorrhagic  con- 
ditions, have  been  found  to  be  associated  with  their  presence ; 


CULTIVATION  OF  THE  BACILLUS 


401 


in  fact,  in  some  cases  the  diphtheritic  lesion  enables  them  to  get 
a  foothold  in  the  tissues,  where 
they  exert  their  usual  action  and 
may  lead  to  extensive  suppurative 
change,  to  septic  poisoning  or  to 
septicaemia.  In  cases  where  a 
gangrenous  process  is  superadded, 
a  great  variety  of  organisms  may 
be  present,  some  of  them  being 
anaerobic.  Against  such  complica- 
tions produced  by  other  organisms 
anti-diphtheritic  serum  produces  no 
favourable  effect. 

Cultivation.  —  The  diphtheria 
bacillus  grows  best  in  cultures  at 
the  temperature  of  the  body ; 
growth  still  takes  place  at  22°  C., 
but  ceases  about  20°  C.  The  best 
media  are  the  following :  Loffler's 
original  medium  (p.  41),  solidified 
blood  serum,  alkaline  blood  serum 
(Lorrain  Smith),  blood  agar,  and  the  ordinary  agar  media.  If 
inoculations  be  made  on  the  surface  of  blood  serum  with  a  piece 

of  diphtheria  membrane, 


a  b 

FIG.  113.  —  Cultures  of  the 
diphtheria  bacillus  on  au 
agar  plate  ;  twenty-six  hours' 
growth.  (Natural  size.) 
(a)  Two  successive  strokes ;  (ft) 
isolated  colonies  from  the  same 
plate. 


colonies   of    the    bacillus 
may    appear    in     twelve 


hours,  and  are  well  formed 
within  twenty-four  hours, 
often  before  any  other 
growths  are  visible.  The 

I  colonies  are  small  circular 

discs  of  opaque  whitish 
R  ^^^  colour,  their  centre  being 
thicker  and  of  darker 
greyish  appearance  when 
viewed  by  transmitted 
light  than  the  periphery. 
Their  margins  are  at  first 
regular,  but  later  they 
become  wavy  or  even 
crenated.  On  the  second 
or  third  day  they  may 

reach  3  mm.  in  size,  but  when  numerous  they  remain  smaller. 

On  the  agar  media  the  colonies  have  much  the  same  appearance 
26 


FIG.  114. — Diphtheria  colonies,  two  days 
old,  on  agar. 


x8. 


402 


DIPHTHERIA 


(Fig.  113)  but  grow  less  quickly,  and  sometimes  they  may  be 

comparatively  minute,  so 
as  rather  to  resemble 
those  of  the  streptococcus 
pyogenes.  In  stroke  cul- 
tures the  growth  forms 
a  continuous  layer  of  the 
same  dull  whitish  colour, 
the  margins  of  which 
often  show  single  colon- 
ies partly  or  completely 
separated.  On  gelatin 
at  22°  C.  a  puncture 
culture  shows  a  line  of 
dots  along  the  needle 
track,  whilst  at  the  sur- 
face a  small  disc  forms, 

FIG.  115.— Diphtheria  bacilli  from  a  twenty-     ra*her     thicker      in     the 
four  hours'  culture  on  agar.  middle.      In  none  of  the 

Stained  with  methylene-blue.     x  1000.          media  does  any  liquefac- 
tion occur.     In  bouillon 

the  organism  produces   a  turbidity  which   soon   settles   to  the 

bottom     and     forms     a 

powdery    layer    on    the 

wall    of    the   vessel.     If 

the  growth  is  started  on 

the  surface  and  the  flask 

is  kept  at  rest,  a  distinct 

scum  forms,  and  this  is 

especially  suitable  for  the 

development     of     toxin. 

Ordinary     bouillon     be-  ^  ^ 

comes    acid    during   the  *-.  /     £tfc 

first  two  or  three   days, 

and    several    days    later 

again  acquires  an  alka- 
line reaction.  If,  how- 
ever, the  bouillon  is 

dextrose-free   (p.  80)   the      FIG.  116. — Diphtheria  bacilli  of  larger  size 

than    in    previous    figure,    showing    also 
irregular  staining  of  protoplasm.      From 
a  three  days'  agar  culture. 
Stained  with  weak  carbol-fuchsin.      x  1000. 


'"im  ~ 


does    not 


acid    reaction 
occur.        The     organism 
not    only   ferments    glu- 
cose, but  also  galactose, 
Isevulose,   maltose,   and    usually  also   glycerine    and    lactose    in 


POWERS  OF  RESISTANCE  OF  BACILLUS       403 

older  cultures;  mannite  and  saccharose  are  not  fermented 
(Graham-Smith). 

In  culture  media  the  bacilli  show  the  same  characters  as  in 
the  membrane,  but  the  beading  is  a  more  marked  feature,  except 
in  the  very  youngest  cultures,  and  sometimes  the  stained  proto- 
plasm has  a  sort  of  septate  appearance  (Figs.  115,  116).  They 
are  at  first  fairly  uniform  in  size  and  shape,  but  later  involution 
forms  may  appear,  especially  on  the  less  favourable  media,  such 
as  agar.  Many  are  swollen  at  their  ends  into  club-shaped  masses 
which  stain  deeply,  and  the  protoplasm  becomes  broken  up  into 
globules  with  unstained  parts  between  (Fig.  117).  Some 
become  thicker  through- 
out, and  segmented  so  *•]  *' 
as  to  appear  like  large  ** 
cocci,  and  others  show  •/ 
globules  at  their  ends,  >^ 
the  rest  of  the  rod 

appearing   as  a   faintly  .-     '-r  „ 

stained  line.     Occasion-      ;  /  ^ 

ally  branched  forms  are  •'      '  -  *^V 

met  with.      The  bacilli  ,1 

are  non-motile,  and  do  -^  «^  / 

not  form  spores.  ~^ 

Staining. — They  take 
up  the  basic  aniline 
dyes,  e.g.  methylene- 
blue  in  watery  solution,  v- 

with  great  readiness,  FIG.  117. — Involution  forms  of  the  diphtheria 
arul  «taii  dppnlv  thp  bacillus;  from  an  agar  culture  of  seven 

PV»    '  days'  growth.     See  also  Plate  III.,  Fig.  13. 

granules  often  giving  the      Stained  with  carbol-thionin-blue.       x  1000. 
metachromatic    reaction 

as  described.  They  also  retain  the  colour  in  Gram's  method, 
though  they  are  more  easily  decolorised  than  the  pyogenic  cocci. 
By  Neisser's  stain  (p.  115)  the  granules  are  stained  almost  black, 
the  rest  of  the  bacillary  substance  yellowish-brown,  or  by  the 
Dew  method,  pink  (Plate  III.,  Fig.  12). 

Powers  of  Resistance,  etc. — In  cultures  the  bacilli  possess 
long  duration  of  life ;  at  room  temperature  they  may  survive 
for  two  months  or  longer.  In  the  moist  condition,  whether  in 
cultures  or  in  membrane,  they  have  a  low  power  of  resistance, 
being  killed  at  60°  C.  in  a  few  minutes.  On  the  other  hand,  in 
the  dry  condition  they  have  great  powers  of  endurance.  In 
membrane  which  is  perfectly  dry,  for  example,  they  can  resist  a 
temperature  of  98°  C.  for  an  hour.  Dried  diphtheria  membrane, 


404  DIPHTHERIA 

kept  in  the  absence  of  light  and  at  the  room  temperature,  has 
been  proved  to  contain  diphtheria  bacilli  still  living  and  virulent 
at  the  end  of  several  months.  The  presence  of  light,  moisture, 
or  a  higher  temperature,  causes  them  to  die  out  more  rapidly. 
Corresponding  results  have  been  obtained  with  bacilli  obtained 
from  cultures  and  kept  on  dried  threads.  These  facts,  especially 
with  regard  to  drying,  are  of  great  importance,  as  they  show  that 
the  contagium  of  diphtheria  may  be  preserved  for  a  long  time 
in  the  dried  membrane. 

Effects  of  Inoculation. — In  considering  the  effects  produced 
in  animals  by  experimental  inoculations  of  pure  cultures,  we 
have  to  keep  in  view  the  local  changes  which  occur  in  diphtheria, 
and  also  the  symptoms  of  general  poisoning. 

As  Loffler  stated  in  his  original  paper,  inoculation  of  the 
healthy  mucous  membranes  of  various  animals  with  pure  cultures 
causes  no  lesion,  but  the  formation  of  false  membrane  may 
result  wrhen  the  surface  is  injured  by  scarification  or  otherwise. 
A  similar  result  may  be  obtained  when  the  trachea  is  inoculated 
after  tracheotomy  has  been  performed.  In  this  case  the 
surrounding  tissues  may  become  the  seat  of  a  blood-stained 
cedema,  and  the  lymphatic  glands  become  enlarged,  the  general 
picture  resembling  pretty  closely  that  of  laryngeal  diphtheria. 
The  membrane  produced  by  such  experiments  is  usually  less 
firm  than  in  human  diphtheria,  and  the  bacilli  in  the  membrane 
are  less  numerous.  Rabbits  inoculated  after  tracheotomy  often 
die,  and  Roux  and  Yersin  were  the  first  to  observe  that  in  some 
cases  paralysis  may  appear  before  death. 

Subcutaneous  injection  in  guinea-pigs  of  diphtheria  bacilli  in 
a  suitable  dose  produces  death  within  thirty-six  hours.  At  the 
site  of  inoculation  there  is  usually  a  small  patch  of  greyish 
membrane,  whilst  in  the  tissues  around  there  is  extensive 
inflammatory  oedema,  often  associated  with  haemorrhages,  and 
there  is  also  some  swelling  of  the  corresponding  lymphatic  glands. 
The  internal  organs  show  general  congestion,  the  suprarenal 
capsules  being  especially  reddened  and  often  haemorrhagic.  The 
renal  epithelium  may  show  cloudy  swelling,  and  there  is  often 
effusion  into  the  pleural  cavities.  After  injection  the  bacilli  in- 
crease in  number  for  a  few  hours,  but  multiplication  soon  ceases, 
and  at  the  time  of  death  they  may  be  less  numerous  than  when 
injected.  The  bacilli  remain  practically  local,  cultures  made 
from  the  blood  and  internal  organs  usually  giving  negative  results, 
though  sometimes  a  few  colonies  may  be  obtained.  If  a  non- 
fatal  dose  of  a  culture  be  injected,  a  local  necrosis  of  the  skin 
and  subcutaneous  tissue  may  follow  at  the  site  of  inoculation. 


THE  TOXINS  OF  DIPHTHERIA  405 

In  rabbits,  after  subcutaneous  inoculation,  results  of  the  same 
nature  follow,  but  these  animals  are  less  susceptible  than  guinea- 
pigs,  and  the  dose  requires  to  be  proportionately  larger.  Roux 
and  Yersin  found  that  after  intravenous  injection  the  bacilli 
rapidly  disappeared  from  the  blood,  and  when  1  c.c.  of  a  broth 
culture  had  been  injected  no  trace  of  the  organisms  could  be 
detected  by  culture  after  twenty-four  hours ;  nevertheless  the 
animals  died  with  symptoms  of  general  toxaemia,  nephritis  also 
being  often  present  (cf.  Cholera,  p.  455).  The  dog  and  sheep 
are  also  susceptible  to  inoculation  with  virulent  bacilli,  but  the 
mouse  and  rat  enjoy  a  high  degree  of  immunity. 

Klein  found  that  cats  also  were  susceptible  to  inoculation.  The 
animals  usually  die  after  a  few  days,  and  post-mortem  there  is  well-marked 
nephritis.  He  also  found  that  after  subcutaneous  injection  in  cows,  a 
vesicular  eruption  appeared  on  the  teats  of  the  udder,  the  fluid  in  which 
contained  diphtheria  bacilli.  At  the  time  of  death  the  diphtheria  bacilli 
were  still  alive  and  virulent  at  the  site  of  injection.  The  most  striking 
result  of  these  experiments  is  that  the  diphtheria  bacilli  passed  into  the 
circulation  and  were  present  in  the  eruption  on  the  udder.  He  considers 
that  this  may  throw  light  on  certain  epidemics  of  diphtheria  in  which 
the  contagion  was  apparently  carried  by  the  milk.  Other  observers, 
e.g.  Abbott,  have,  however,  failed  to  obtain  similar  results.  Dean  and 
Todd,in  investigating  an  outbreak  of  diphtheria  traceable  to  a  milk  supply, 
found  a  vesicular  eruption  on  the  teats  of  the  udder  in  which  diphtheria 
bacilli  were  present.  They,  however,  came  to  the  conclusion  that  these 
bacilli  were  not  the  cause  of  the  eruption,  but  were  the  result  of  a 
secondary  contamination,  probably  from  the  saliva  of  the  milkers.  The 
existence  of  a  true  diphtheria  infection  in  cows  must  still  be  considered 
doubtful.  A  case  of  true  diphtheria  in  the  horse  has  been  described  by 
Cobbett. 

The  Toxins  of  Diphtheria. — As  in  the  above  experiments 
the  symptoms  of  poisoning  and  ultimately  a  fatal  result  occur 
when  the  bacilli  are  diminishing  in  number,  or  even  after  they 
have  practically  disappeared,  Roux  and  Yersin  inferred  that  the 
chief  effects  were  produced  by  toxins,  and  this  supposition  they 
proved  to  be  correct.  They  showed  that  broth  cultures  of  three 
or  four  weeks'  growth  freed  from  bacilli  by  filtration  were  highly 
toxic.  The  filtrate  when  injected  into  guinea-pigs  and  other 
animals  produces  practically  the  same  effects  as  the  living  bacilli ; 
locally  there  is  fibrinous  exudation  but  a  considerable  amount 
of  inflammatory  (jedema,  and,  if  the  animal  survive  long  enough, 
necrosis  in  varying  degree  of  the  superficial  tissues  may  follow. 
The  toxicity  may  be  so  great  that  '005  c.c.  or  even  less  may  be 
fatal  to  a  guinea-pig  in  five  days. 

After  injection  either  of  the  toxin  or  of  the  living  bacilli, 
\\lieii  the  animals  survive  long  enough,  paralytic  phenomena 


406  DIPHTHERIA 

occasionally  occur.  The  hind-limbs  are  usually  affected  first,  the 
paralysis  afterwards  extending  to  other  parts,  though  sometimes 
the  fore-limbs  and  neck  first  show  the  condition.  Sometimes 
symptoms  of  paralysis  do  not  appear  till  two  or  three  weeks 
after  inoculation.  After  paralysis  has  appeared,  a  fatal  result 
usually  follows  in  the  smaller  animals,  but  in  dogs  recovery  may 
take  place.  There  is  evidence  that  these  paralytic  phenomena  are 
produced  by  toxone,  as  they  specially  occur  when  there  is  injected 
along  with  the  toxin  sufficient  antitoxin  to  neutralise  the  more 
rapidly  acting  toxin  proper.  This  toxone  is  supposed  by  Ehrlich 
to  have  a  different  toxic  action,  i.e.  a  different  toxophorous 
group  (p.  198),  from  that  of  the  ordinary  toxin  ;  it  produces 
the  late  nervous  phenomena,  while  its  local  action  on  the  tissues 
is  very  slight.  It  also  has  a  weaker  affinity  for  antitoxin,  and 
thus  much  of  it  may  be  left  unneutralised.  It  is  to  be  noted  in 
this  connection  that  paralytic  symptoms  are  of  not  uncommon 
•  occurrence  in  the  human  subject  after  treatment  with  antitoxin, 
the  explanation  of  which  occurrence  is  probably  the  same  as 
that  just  given.  One  point  of  much  interest  is  the  high  degree 
of  resistance  to  the  toxin  possessed  by  mice  and  rats.  Roux  and 
Yersin,  for  example,  found  that  2  c.c.  of  toxin,  which  was 
sufficient  to  kill  a  rabbit  in  sixty  hours,  had  no  effect  on  a 
mouse,  whilst  of  this  toxin  even  —  c.c.  produced  extensive 
necrosis  of  the  skin  of  the  guinea-pig. 

Preparation  of  the  Toxin. — The  obtaining  of  a  very  active 
toxin  in  large  quantities  is  an  essential  in  the  preparation  of  anti- 
diphtheritic  serum.  Certain  conditions  favour  the  development 
of  a  high  degree  of  toxicity,  namely,  a  free  supply  of  oxygen,  the 
presence  of  a  large  proportion  of  peptone  or  albumin  in  the 
medium,  and  the  absence  of  substances  which  produce  an  acid 
reaction.  In  the  earlier  work  a  current  of  sterile  air  was 
made  to  pass  over  the  surface  of  the  medium,  as  it  was  found 
that  by  this  means  the  period  of  acid  reaction  was  shortened  and 
the  toxin  formation  favoured.  This  expedient  is  now  considered 
unnecessary  if  an  alkaline  medium  free  from  glucose  is  used,  as 
in  this  no  acid  reaction  is  developed ;  it  is  then  sufficient  to 
grow  the  cultures  in  shallow  flasks.  The  absence  of  glucose 
may  be  attained  by  the  method  described  above  (p.  80),  or  by 
using  for  the  preparation  of  the  meat  extract  flesh  which  is  just 
commencing  to  putrefy  (Spronck).  L.  Martin  uses  a  medium 
composed  of  equal  parts  of  freshly  prepared  peptone  (by  digest- 
ing pigs'  stomachs  with  HC1  at  35°  C.),  and  glucose-free  veal 
bouillon.  By  this  medium  he  has  obtained  a  toxin  of  which 
c.c.  is  the  fatal  dose  to  a  guinea-pig  of  500  grms.  Park 


NATURE  OF  THE  TOXIN  407 

and  Williams  and  also  Dean  find  that  the  amount  of  glucose 
present  in  ordinary  beef  is  not  sufficient  to  interfere  with  toxin 
formation,  provided  that  a  considerable  amount  of  peptone,  2  per 
cent.,  be  added,  and  the  medium  be  made  sufficiently  alkaline ; 
after  making  it  neutral  to  litmus  they  add  to  each  litre  of  broth 
7  c.c.  of  normal  caustic  soda  solution.  There  is  in  all  cases  a 
period  at  which  the  toxicity  reaches  a  maximum ;  Roux  and 
Yersin  found  this  period  to  be  two  to  three  weeks,  but  later 
observers  find  that  in  favourable  conditions  the  greatest  toxicity 
is  reached  about  the  tenth  to  twelfth  day,  sometimes  even 
earlier.  It  may  be  added  that  the  power  of  toxin  formation 
varies  much  in  different  races  of  the  diphtheria  bacillus,  and 
that  many  may  require  to  be  tested  ere  one  suitable  is 
obtained. 

Properties  and  Nature  of  the  Toxin. — The  toxic  substance  in 
filtered  cultures  is  a  relatively  unstable  body.  When  kept  in 
sealed  tubes  in  the  absence  of  light,  it  may  preserve  its  powers 
little  altered  for  several  months,  but,  on  the  other  hand,  it 
gradually  loses  them  when  exposed  to  the  action  of  light  and 
air.  As  will  be  shown  later  (p.  527),  the  toxin  probably  does 
not  become  destroyed,  but  its  toxophorous  group  suffers  a  sort  of 
deterioration,  so  that  a  toxoid  is  formed  which  has  still  the 
power  of  combining  with  antitoxins.  Heating  at  58°  C.  for 
two  hours  destroys  the  toxic  properties  in  great  part,  but  not 
altogether.  When,  however,  the  toxin  is  evaporated  to  dryness, 
it  has  much  greater  resistance  to  heat.  One  striking  fact, 
discovered  by  Roux  and  Yersin,  is  that  after  an  organic  acid, 
such  as  tartaric  acid,  is  added  to  the  toxin  the  toxic  property 
disappears,  but  it  can  be  in  great  part  restored  by  again 
making  the  fluid  alkaline. 

Guinochet  found  that  toxin  was  formed  by  the  bacilli  when 
grown  in  urine  with  no  proteid  bodies  present.  After  growth 
had  taken  place  he  could  not  detect  proteid  bodies  in  the  fluid, 
but,  on  account  of  the  very  minute  amount  of  toxin  present, 
their  absence  could  not  be  excluded.  Uschinsky  also  found  that 
toxic  bodies  were  produced  by  diphtheria  bacilli  when  grown  in 
a  proteid-free  medium.1  It  follows  from  this  that  if  the  toxin 
is  a  proteid,  it  may  be  formed  by  synthesis  within  the  bodies  of 
the  bacilli.  Brieger  and  Boer  have  separated  from  diphtheria 
cultures  a  toxic  body  which  gives  no  proteid  reaction  (vide  p. 

i  Uschinsky's  medium  has  the  following  composition  :  water,  1000  parts  ; 
glycerin,  30-40  ;  sodium  chloride,  5-7  ;  calcium  chloride,  'I  ;  magnesium 
sulphate,  '2- '4  ;  di-potassium  phosphate,  *2-'25 ;  ammonium  lactate,  6-7  ; 
sodium  asparaginate,  3-4. 


408  DIPHTHERIA 

193).  Whether  or  not  diphtheria  toxin  is  of  proteid  nature 
must,  however,  be  considered  to  be  a  question  not  yet  settled. 

Toxic  bodies  have  also  been  obtained  from  the  tissues  of  those 
who  have  died  from  diphtheria.  Roux  and  Yersin,  by  using 
a  filtered  watery  extract  from  the  spleen  from  very  virulent  cases 
of  diphtheria,  produced  in  animals  death  after  wasting  and 
paralysis,  and  also  obtained  similar  results  by  employing  the 
urine.  The  subject  of  toxic  bodies  in  the  tissues  has,  however, 
been  specially  worked  out  by  Sidney  Martin.  He  has  separated 
from  the  tissues,  and  especially  from  the  spleen,  of  patients  who 
have  died  from  diphtheria,  by  precipitation  with  alcohol,  chemical 
substances  of  two  kinds,  namely,  albumoses  (proto-  and  deutero-, 
but  especially  the  latter),  and  an  organic  acid.  The  albumoses, 
when  injected  into  rabbits,  especially  in  repeated  doses,  produce 
fever,  diarrhoea,  paresis,  and  loss  of  weight,  with  ultimately  a 
fatal  result.  He  further  found  that  this  paresis  is  due  to 
well-marked  changes  in  the  nerves.  Substances  obtained  from 
diphtheria  membrane  have  an  action  like  that  of  the  bodies 
obtained  from  the  spleen,  but  in  higher  degree.  Martin  con- 
siders that  this  is  due  to  the  presence  in  the  membrane  of  an 
enzyme  which  has  a  proteolytic  action  within  the  body,  resulting 
in  the  formation  of  poisonous  albumoses. 

Immunity. — This  is  described  in  the  general  chapter  on 
Immunity.  It  is  sufficient  to  state  here  that  a  high  degree  of 
immunity,  against  both  the  bacilli  and  their  toxins,  can  be 
produced  in  various  animals  by  gradually  increasing  doses  either 
of  the  bacilli  or  of  their  filtered  toxins  (vide  Chapter  XXI.). 

Variations  in  the  Virulence  of  the  Diphtheria  Bacillus. — In 
cultures  on  serum  the  diphtheria  bacilli  retain  their  virulence 
fairly  well,  but  they  lose  it  much  more  quickly  on  less  suitable 
media,  such  as  glycerin  agar.  Roux  and  Yersin  found  that, 
when  the  bacilli  were  grown  at  an  abnormally  high  temperature, 
namely,  39*5°  C.,  and  in  a  current  of  air,  the  virulence  diminished 
so  much  that  they  became  practically  innocuous.  When  the 
virulence  was  much  diminished,  these  observers  found  that  it 
could  be  restored  if  the  bacilli  were  inoculated  into  animals 
along  with  streptococci,  inoculation  of  the  bacilli  alone  not 
being  successful  for  this  purpose.  If,  however,  the  virulence 
had  fallen  very  low,  even  the  presence  of  the  streptococci  was 
insufficient  to  restore  it.  The  virulence  is  tested  by  the  amount 
of  living  bacilli  necessary  to  produce  a  fatal  result  on  injection, 
and  is  to  be  distinguished  from  the  power  of  producing  toxin  in 
a  fluid  medium;  as  pointed  out  by  Dean,  the  two  properties 
often  do  not  correspond.  It  has  been  abundantly  established 


BACILLI  ALLIED  TO  DIPHTHERIA  BACILLUS     409 

that,  after  the  cure  of  the  disease,  the  bacilli  may  persist  in  the 
mouth  for  weeks,  though  they  often  quickly  disappear.  Roux 
and  Yersin  found,  by  making  cultures  at  various  stages  after 
the  termination  of  the  disease,  that  these  bacilli  in  the  mouth 
gradually  become  attenuated. 

I  j,  Martin,  moreover,  has  shown  that  some  races  of  diphtheria 
bacillus  are  so  attenuated  that  1  c.c.  of  a  twenty-four  hours' 
growth  in  bouillon  does  not  cause  death  in  a  guinea-pig,  yet  their 
true  nature  is  shown  not  only  by  their  miscroscopical  characters, 
etc.,  but  also  by  the  fact  that  on  more  prolonged  growth  they 
form  small  quantities  of  toxin,  which  is  neutralisable  by  diphtheria 
antitoxin.  The  persistence  of  these  non-virulent  bacilli  in  the 
throats  of  those  who  have  suffered  from  the  disease,  and  their 
occasional  presence  in  quite  healthy  individuals,  may  manifestly 
be  of  importance  in  relation  to  the  continuance  of  the  infection 
and  the  reappearance  of  epidemics  of  the  disease. 

BACILLI  ALLIED  TO  THE  DIPHTHERIA  BACILLUS. 

Bacteriological  examinations  carried  on  within  recent  times 
have  shown  that  the  diphtheria  bacillus  is  merely  a  member  of 
a  group  of  organisms  with  closely  allied  characters  which  are 
of  common  occurrence  and  have  a  wide  distribution.  The  terms 
"pseudo-diphtheria  bacilli "  and  "diphtheroid  bacilli "  have  been 
applied  in  a  loose  way  to  organisms  which  resemble  the 
diphtheria  bacillus  microscopically,  especially  as  regards  the 
beaded  appearance.  Such  bacilli  have  been  obtained  from  the 
mouth,  nose,  skin,  genital  organs,  and  even  from  the  blood  in 
certain  diseases.  They  are  to  be  met  with  sometimes  in  condi- 
tions of  health,  and  they  have  been  obtained  from  many  diverse 
morbid  conditions  —  from  skin  diseases,  from  coryza,  from 
leprosy,  and  even  from  general  paralysis  of  the  insane.  As 
has  been  found  with  other  groups,  the  differentiation  is  a  matter 
of  considerable  difficulty.  Some  are  practically  identical  with 
the  diphtheria  bacillus  both  morphologically  and  culturally,  and 
a  few  even  give  the  characteristic  reaction  with  Neisser's  stain ; 
others,  again,  differ  in  essential  particulars.  The  fermentative 
action  on  sugars1  has  also  been  called  into  requisition  as  a 
means  of  distinguishing  them,  but  the  results  obtained  cannot 
be  said  to  be  of  a  definite  character,  and  further  work  is 
necessary.  It  may  be  stated,  however,  that  most  observers 
have  found  the  diphtheria  bacillus  of  all  the  members  of  the 
group  to  'be  the  most  active  acid-producer,  though  here  the 
1  Vide  a  paper  by  Graham-Smith,  Journal  oj  Hygiene,  vi.  286. 


410  DIPHTHERIA 

difference  seems  to  be  one  of  degree  rather  than  of  kind.  The 
absence  of  the  power  of  fermenting  certain  sugars,  notably 
glucose,  may,  however,  be  accepted  in  any  particular  case  as 
sufficient  to  exclude  the  organism  from  being  the  diphtheria 
bacillus.  From  these  facts,  and  from  what  has  been  stated 
with  regard  to  attenuated  diphtheria  bacilli,  it  will  be  seen  that 
an  absolute  decision  as  to  the  nature  of  a  suspected  organism 
may  in  some  cases  be  a  practical  impossibility.  It  may  be  that 
some  of  the  "  diphtheroid "  organisms  cultivated  have  really 
been  non-virulent  diphtheria  bacilli.  The  bearing  of  this  on 
the  practical  means  of  diagnosis  will  be  discussed  below. 

Ford  Robertson  and  his  co-workers  have  obtained  from 
numerous  cases  of  general  paralysis  of  the  insane  cultures  of  a 
diphtheroid  organism,  which  he  considers  is  the  chief  agent  in 
producing  the  condition  of  chronic  intoxication  underlying  the 
disease.  The  organism  has  been  obtained  from  various  situations, 
including  the  central  nervous  system,  but  it  seems  to  flourish 
specially  in  the  respiratory  and  alimentary  tracts.  It  closely 
resembles  the  diphtheria  bacillus  ;  the  morphological  and  cultural 
characters  are  indeed  practically  identical,  but  the  diphtheroid 
bacillus  is  non-pathogenic  to  the  guinea-pig.  Robertson  and 
Shennan  found  that  when  administered  to  rats  by  the  alimentary 
tract  it  produced  certain  nervous  symptoms  which  were  associ- 
ated with  changes  in  the  brain  of  the  same  order  as  those  in 
general  paralysis.  Further  research  on  this  subject  is  still 
necessary. 

The  term  "  pseudo-diphtheria  bacillus  "  is  often  restricted  by 
present  writers  to  an  organism  frequently  met  with  in  the 
throat.  This  organism,  which  is  also  known  as  Hofmann's 
bacillus,  merits  a  separate  description. 

Hofmann's  Bacillus  —  Pseudo-Diphtheria  Bacillus.  —  This 
organism,  described  by  Hofmann  in  1888,  is  probably  the  same 
as  one  observed  by  Loffler  in  the  previous  year,  and  regarded 
by  him  as  being  a  distinct  species  from  the  diphtheria  bacillus. 
The  organism  is  a  shorter  bacillus  than  the  diphtheria  bacillus, 
with  usually  a  single  unstained  septum  running  across  it,  though 
sometimes  there  may  be  more  than  one  (Fig.  118).  The  typical 
beaded  appearance  is  rarely  seen,  and  the  characteristic  reaction 
with  Neisser's  stain  is  not  given,  though  in  old  cultures  a  few 
granules  which  stain  deeply  may  sometimes  be  found.  It  grows 
readily  on  the  same  media  as  the  diphtheria  bacillus,  but  the 
colonies  are  whiter  and  more  opaque.  It  does  not  form  acid 
from  glucose  or  other  sugars,  and  is  non-pathogenic  to  the 
guinea-pig.  Involution  forms  may  sometimes  be  produced  by 


PSEUDO-DIPHTHERIA  BACILLUS  411 

it.  It  is  usually  a  relatively  easy  matter  to  distinguish  this 
organism  from  the  diphtheria  bacillus. 

Hofmann's  bacillus  is  of  comparatively  common  occurrence  in 
the  throat  in  normal  as  well  as  diseased  conditions,  including 
diphtheria  ;  it  seems  to  be  specially  frequent  in  poorly  nourished 
children  of  the  lower  classes.  Cobbett  found  it  157  times  in  an 
examination  of  692  persons  examined,  of  whom  650  were  not 
suffering  from  diphtheria.  Boycott's  statistics  show  that  the 
time  of  its  maximum  seasonal  prevalence  precedes  that  of  the 
diphtheria  bacillus.  To  what  extent,  if  any,  it  is  responsible 
for  pathological  changes  in  the  throat,  must  be  considered  a 
question  which  is  not 
yet  settled.  Hewlett 
and  Knight  have  found 
evidence  that  a  true 
diphtheria  bacillus  may 
assume  the  characters  of 
Hofmann's  bacillus,  but 
attempts  to  effect  the 
transformation  have  met 
with  negative  results  in 
the  hands  of  other 
observers.  The  general 
opinion  is  that  the  two 
organisms  are  distinct 
species  with  compara- 
tively  easily  distinguished 

characters.  Flf;  ns.—Pseudo-diphtheria  bacillus  (Hof- 

mann's).    Young  agar  culture.     See  also 
Xerosis  Bacillus.  —  This          piate  III.,  Fig.  14. 

term  has  been  given  to  an  Stained  with  thionin-blue.     x  1000. 

organism   first  observed  by 
Kuschbert   and    Neisser    in 

xerosis  of  the  conjunctiva,  and  which  has  been  since  found  in  many  other 
affections  of  the  conjunctiva  and  also  in  normal  conditions.  Morpho- 
logically it  is  practically  similar  to  the  diphtheria  bacillus,  and  even  in 
cultures  presents  very  minor  differences  ;  it,  however,  grows  more  slowly 
on  serum,  and  its  colonies  have  a  tougher  consistence  and  a  more  irregular 
margin.  It  is  non-virulent  to  animals,  and  does  not  produce  an  acid 
reaction  in  glucose  bouillon,  or  does  so  to  only  a  slight  extent ;  in  this 
way  it  can  be  distinguished  from  the  diphtheria  bacillus.  It  is  still 
doubtful  whether  it  is  pathogenic  to  the  human  subject.  Its  morpho- 
logical characters  are  shown  in  Fig.  119. 

Action  of  the  Diphtheria  Bacillus  —  Summary. — From  a 
study  of  the  morbid  changes  in  diphtheria  and  of  the  results 
produced  experimentally  by  the  bacillus  and  its  toxins,  the 


412  DIPHTHERIA 

following  summary  may  be  given  of  its  action  in  the  body. 
Locally,  the  bacillus  produces  inflammatory  change  with 
fibrinous  exudation,  but  at  the  same  time  cellular  necrosis  is 
also  an  outstanding  feature.  Though  false  membranes  have  not 
been  produced  by  the  toxins,  a  necrotic  action  may  result  when 
these  are  injected  subcutaneously.  The  toxins  also  act  upon 
the  blood  vessels,  and  hence  oedema  and  tendency  to  hemorrhage 
are  produced ;  this  action  on  the  vessels  is  also  exemplified  by 
the  general  congestion  of  organs.  The  hyaline  change  in  the 
walls  of  arterioles  and  capillaries  so  often  met  with  in  diphtheria 
is  another  example  of  the  action  of  the  toxin.  The  toxins  have 
also  a  pernicious  action  on  highly  developed  cells  and  on  nerve 

fibres.    Thus  in  the  kidney 
g.    ^  cloudy     swelling     occurs, 

\        "•**^*iW»  which    may   be    followed 

by  actual  necrosis  of  the 
*tjj£  secreting  cells,  and  along 

with  these  changes  albu- 
minuria  is  present.  The 
action  is  also  well  seen  in 
the  case  of  the  muscle 
fibres  of  the  heart,  which 


„ 


V>*%  m&y   undergo    a    sort    of 

hyaline  change,  followed 
by  granular  disintegra- 
tion or  by  an  actual 

- "        ;  fatty  degeneration.    These 

FIG.  119.— Xerosis  bacillus  from  a  young       changes      are      of      great 
agar  culture.     xlOOO.  importance  in  relation  to 

heart  failure  in  the  disease. 

Changes  of  a  somewhat  similar  nature  have  been  recently 
observed  in  the  nerve  cells  of  the  central  nervous  system,  those 
lying  near  the  capillaries,  it  is  said,  being  affected  first.  There 
is  also  the  striking  change  in  the  peripheral  nerves,  which  is 
shown  first  by  the  disintegration  of  the  medullary  sheaths  as 
already  described.  It  is,  however,  still  a  matter  of  dispute  to 
what  extent  these  nerve  lesions  are  of  primary  nature  or 
secondary  to  changes  in  the  nerve  cells. 

Methods  of  Diagnosis.  —  The  bacteriological  diagnosis  of 
diphtheria  depends  on  the  discovery  of  the  bacillus.  As  the 
bacillus  occurs  in  largest  numbers  in  the  membrane,  a  portion  of 
this  should  be  obtained  whenever  it  is  possible,  and  transferred 
to  a  sterile  test-tube.  (The  tube  can  be  readily  sterilised  by 
boiling  some  water  in  it.)  If,  however,  membrane  cannot  be 


METHODS  OF  DIAGNOSIS  413 

obtained,  a  scraping  of  the  surface  with  a  platinum  loop  may  be 
sufficient.  Where  the  membrane  is  confined  to  the  trachea  the 
bacilli  are  often  present  in  the  secretions  of  the  pharynx,  and 
may  be  obtained  from  that  situation  by  swabbing  it  with  cotton- 
wool (non-antiseptic),  the  swab  being  put  into  a  sterile  tube  or 
bottle  for  transport.  A  convenient  method  is  to  twist  a  piece  of 
cotton-wool  round  the  roughened  end  of  a  piece  of  very  stout 
iron  wire,  6  inches  long,  and  pass  the  other  end  of  the  latter 
through  a  cotton  plug  inserted  in  the  mouth  of  a  test-tube 
(compare  Fig.  46,  the  wire  taking  the  place  of  the  pipette),  and 
sterilise.  In  use  the  wire  and  plug  are  extracted  in  one  piece, 
and  after  swabbing  are  replaced  in  the  tube  for  transit.  A 
scraping  may  be  made  off  the  swab  for  microscopic  examination, 
and  the  swab  may  be  smeared  over  the  surface  of  a  serum  tube 
to  obtain  a  culture.  This  method  of  taking  and  treating  swabs 
is  that  usually  employed  in  routine  public  health  work.  The 
results  obtained  ordinarily  suffice  for  the  diagnosis  of  cases 
suspected  to  be  diphtheritic  in  nature. 

The  means  for  identifying  the  bacillus  are  (a)  By  Micro- 
scopical Examination.  —  For  microscopical  examination  it  is 
sufficient  to  tease  out  a  piece  of  the  membrane  with  forceps  and 
rub  it  on  a  cover-glass ;  if  it  be  somewhat  dry,  a  small  drop  of 
normal  saline  should  be  added.  The  films  are  then  dried  in  the 
usual  way,  and  stained  with  any  ordinary  basic  stain,  though 
methylene-blue  is  on  the  whole  to  be  preferred,  used  either  as 
a  saturated  watery  solution  or  in  the  form  of  Loffler's  solution. 
After  staining  for  two  or  three  minutes,  the  films  are  washed 
in  water,  dried,  and  mounted.  As  a  rule  no  decolorising  is 
necessary,  as  the  blue  does  not  overstain.  Neisser's  stain  (p.  1 1 5) 
may  also  be  used  with  advantage,  although  it  is  to  be  noted 
that  sometimes  in  a  secretion  the  diphtheria  bacillus  does  not 
react  typically  to  this  stain.  Any  secretion  from  the  pharynx 
or  other  part  is  to  be  treated  in  the  same  way.  The  value  of 
microscopical  examination  alone  depends  much  upon  the  experi- 
ence of  the  observer.  In  some  cases  the  bacilli  are  present  in 
characteristic  form  in  such  numbers  as  to  leave  no  doubt  in 
the  matter.  In  other  cases  a  few  only  may  be  found,  mixed 
with  large  numbers  of  other  organisms,  and  sometimes  their 
characters  are  not  sufficiently  distinct  to  render  a  definite  opinion 
possible.  The  bacillus  may  be  frequently  obtained  by  means 
of  cultures,  when  the  result  of  microscopical  examination  is 
inconclusive.  As  already  said,  however,  microscopical  examina- 
tion alone  is  more  reliable  after  the  observer  has  had  experience 
in  examining  cases  of  diphtheria  and  making  cultures  from  them. 


414  DIPHTHERIA 

(6)  By  making  Cultures. — For  this  purpose  a  piece  of  the 
membrane  should  be  separated  by  forceps  from  the  pharynx  or 
other  part  when  that  is  possible.  It  should  be  then  washed 
well  in  a  tube  containing  sterile  water,  most  of  the  surface  im- 
purities being  removed  in  this  way.  A  fragment  is  then  fixed  in 
a  platinum  loop  by  means  of  sterile  forceps,  and  a  series  of 
stroke  cultures  is  made  on  the  surface  of  any  of  the  media 
mentioned  (p.  401),  the  same  portion  of  the  membrane  being 
always  brought  into  contact  with  the  surface.  The  tubes  are 
then  incubated  at  37°  C.,  and,  in  the  case  of  the  serum 
media  and  blood-agar,  the  circular  colonies  of  the  diphtheria 
bacillus  are  well  formed  within  twenty -four  hours.  A  small 
portion  of  a  colony  is  then  removed  by  means  of  a  platinum 
needle,  stained,  and  examined  in  the  usual  way,  Neisser's  stain 
being  also  applied.  When  the  material  has  been  taken  from 
the  throat,  an  organism  with  all  the  morphological  and  cultural 
characters  of  the  diphtheria  bacillus  may  for  all  practical 
purposes  be  accepted  as  the  diphtheria  bacillus. 

In  cases  where  a  suspicion  arises  that  the  organism  found 
is  a  '  pseudo  -  diphtheria  bacillus,  bouillon  containing  a  trace 
of  glucose  should  be  inoculated  and  incubated  at  37°  C. 
The  reaction  should  be  tested  after  one  and  after  two  days' 
growth.  If  it  remains  alkaline,  the  diphtheria  bacillus  may  be 
excluded.  If  an  acid  reaction  results,  then  all  the  microscopical 
and  cultural  characters  must  be  carefully  observed,  and  the 
virulence  of  the  bacillus  may  be  ascertained  by  inoculating  a 
guinea-pig,  say  with  1  c.c.  of  a  broth  culture  of  two  days'  growth. 
(See  also  pp.  404,  410.)  A  fatal  result  with  characteristic 
appearances  may  be  taken  as  positive  evidence  ;  but  if  the  animal 
survive  there  is  still  theoretically  the  possibility  that  the 
organism  is  an  attentuated  diphtheria  bacillus  (p.  408). 


CHAPTER  XVII. 

TETANUS1:  CONDITIONS  CAUSED  BY  OTHER 
ANAEROBIC  BACILLI. 

Introductory.  —  Tetanus  (German,  Wundstarrkrampf)  is  a 
disease  which  in  natural  conditions  affects  chiefly  man  and  the 
horse.  Clinically  it  is  characterised  by  the  gradual  onset  of 
general  stiffness  and  spasms  of  the  voluntary  muscles,  com- 
mencing in  those  of  the  jaw  and  the  back  of  the  neck,  and 
extending  to  all  the  muscles  of  the  body.  These  spasms 
are  of  a  tonic  nature,  and,  as  the  disease  advances,  succeed 
each  other  with  only  a  slight  intermission  of  time.  There 
are  often,  towards  the  end  of  a  case,  fever  and  rise  of 
respiration  and  pulse-rate.  The  disease  is  usually  associated 
with  a  wound  received  from  four  to  fourteen  days  previously, 
and  which  has  been  denied  by  earth  or  dung.  The  disease  is, 
in  the  majority  of  cases,  fatal. 

Historical. — The  general  association  of  the  development  of  tetanus  with 
the  presence  of  wounds-,  though  these  might  be  very  small,  suggested  that 
some  infection  took  place  through  the  latter,  but  for  long  nothing  was 
known  as  to  the  nature  of  this  infection.  Carle  and  Rattone  in  1884 
announced  that  they  had  produced  the  disease  in  a  number  of  animals  by 
inoculation  with  material  from  a  wound  in  tetanus.  They  thus  demon- 
strated the  transmissibility  of  the  disease.  Nicolaier  (1885)  infected  mice 
and  rabbits  with  garden  earth,  and  found  that  many  of  them  developed 
tetanus.  Suppuration  occurred  in  the  neighbourhood  of  the  point  of 
inoculation,  and  in  this  pus,  besides  other  organisms,  there  was  always 
present,  when  tetanus  had  occurred,  a  bacillus  having  certain  constant 
microscopic  characters.  Inoculation  of  fresh  animals  with  such  pus 
reproduced  the  disease.  Nicolaier's  attempts  at  its  isolation  by  the 
ordinary  gelatin  plate-culture  method  were,  however,  unsuccessful.  He 
succeeded  in  getting  it  to  grow  in  liquid  blood  serum,  but  always  in 

1  This  disease  is  not  to  be  confused  with  the  "  tetany  "  of  infants,  which  in 
its  essential  pathology  probably  differs  from  tetanus  (vide  Frankl-Hochwart, 
"  Die  Tetanic  der  Erwachsenen,"  Vienna,  1907).  This  remark  of  course  does 
not  exclude  the  possibility  of  the  occurrence  of  true  tetanus  in  very  young 
subjects. 

415 


416  TETANUS 

mixture  with  other  organisms.  Infection  of  animals  with  such  a  culture 
produced  the  disease.  These  results  were  confirmed  by  Rosenbach,  who, 
though  failing  to  obtain  a  pure  culture,  cultivated  the  other  organisms 
present,  and  inoculated  them,  but  with  negative  results.  He  further 
pointed  out,  as  characteristic  of  the  bacillus,  its  development  of  terminal 
spores.  In  1889,  Kitasato  succeeded  in  isolating  from  the  local  suppura- 
tion of  mice  inoculated  from  a  human  case,  several  bacilli,  only  one  of 
which,  when  injected  in  pure  culture  into  animals,  caused  the  disease, 
and  which  was  now  named  the  b.  tetani.  This  organism  is  the  same  as 
that  observed  by  Nicolaier  and  Rosenbach.  Kitasato  found  that  the 
cause  of  earlier  culture  failures  was  the  fact  that  it  could  only  grow  in  the 
absence  of  oxygen.  The  pathology  of  the  disease  was  further  elucidated 
by  Faber,  who,  having  isolated  bacterium- free  poisons  from  cultures, 
reproduced  the  symptoms  of  the  disease. 

Bacillus  Tetani. — If  in  a  case  of  tetanus  naturally  arising 
in  man,  there  be  a  definite  wound  with  pus  formation  or  necrotic 
change,  the  bacillus  tetani  may  be  recognised  in  film  preparations 
from  the  pus,  if  the  characteristic  spore  formation  has  occurred 
(Fig.  120).  If,  however,  the  tetanus  bacilli  have  not  formed 
spores,  they  appear  as  somewhat  slender  rods,  without  present- 
ing any  characteristic  features.  There  is  usually  present  in  such 
pus  a  great  variety  of  other  organisms — cocci  and  bacilli.  The 
characters  of  the  bacillus  are,  therefore,  best  studied  in  cultures. 
It  is  then  seen  to  be  a  slender  organism,  usually  about  4  /JL  to 
5  JJL  in  length  and  *4  JJL  in  thickness,  with  somewhat  rounded 
ends.  Besides  occurring  as  short  rods  it  also  develops 
filamentous  forms,  the  latter  being  more  common  in  fluid  media. 
It  stains  readily  by  any  of  the  usual  stains  and  also  by  Gram's 
method.  A  feature  in  it  is  the  uniformity  with  which  the 
protoplasm  stains.  It  is  very  slightly  motile,  and  its  motility 
can  be  best  studied  in  an  anaerobic  hanging-drop  preparation. 
When  stained  by  the  special  methods  already  described, 
it  is  found  to  possess  numerous  delicate  flagella  attached 
both  at  the  sides  and  at  the  ends  (Fig.  121).  These  flagella, 
though  they  may  be  of  considerable  length,  are  usually 
curled  up  close  to  the  body  of  the  bacillus.  The  formation  of 
flagella  can  be  best  studied  in  preparations  made  from  surface 
anaerobic  cultures  (p.  68).  As  is  the  case  with  many  other 
anaerobic  flagellated  bacteria,  the  flagella,  on  becoming  detached, 
often  become  massed  together  in  the  form  of  spirals  of  striking 
appearance  (Fig.  122).  At  incubation  temperature  b.  tetani 
readily  forms  spores,  and  then  presents  a  very  characteristic 
appearance.  The  spores  are  round,  and  in  diameter  may  be 
three  or  four  times  the  thickness  of  the  bacilli.  They  are 
developed  at  one  end  of  a  bacillus,  which  thus  assumes  what  is 
usually  described  as  the  drumstick  form  (Figs.  120,  123).  In 


BACILLUS  TETANI 


41 


a  specimen  stained  with  a  watery  solution  of  gentian-violet  or 
methylene-blue,  the  spores  are  uncoloured  except  at  the  periphery, 
so  that  the  appearance  of  a  small  ring  is  produced  ;  if  a  powerful 
stain  such  as  carbol-fuchsin  be  applied  for  some  time,  the  spores 
become  deeply  coloured  like  the  bacilli.  Further,  especially  if 


^ 


Fio.  120.  —  Film  preparation  of  discharge  from  wound  in  a  case 
of  tetanus,  showing  several  tetanus  bacilli  of  "drumstick"  form. 
(The  thicker  bacillus  present  is  not  a  tetanus  bacillus,  but  a 
putrefactive  anaerobe  which  was  obtained  in  pure  culture  from  the 
wound.  ) 

Stained  with  gentian  -violet,     x  1000. 


the  preparation  be  heated,  many  spores  may  become  free  from  the 
bacilli  in  which  they  were  formed. 

Isolation.  —  The  isolation  of  the  tetanus  bacillus  is  somewhat 
difficult.  By  inoculation  experiments  in  animals,  its  natural 
habitat  has  been  proved  to  be  garden  soil,  and  especially  the 
contents  of  dung-heaps,  where  it  probably  leads  a  saprophytic 
existence,  though  its  function  as  a  saprophyte  is  unknown.  From 
such  sources  and  from  the  pus  of  wounds  in  tetanus,  occurring 
naturally  or  experimentally  produced,  it  has  been  isolated^by 

27 


418 


TETANUS 


means  of  the  methods  appropriate  for  anaerobic  bacteria.     The 
best  methods  for  dealing  with  such  pus  are  as  follows  : — 

(1)  The  principle  is  to  take  advantage  of  the  resistance  of  the 
spores  of  the  bacillus  to  heat.  A  sloped  tube  of  inspissated 
serum  or  a  deep  tube  of  glucose  agar  is  inoculated  with  the  pus 
and  incubated  anaerobically  at  37°  C.  for  forty-eight  hours,  at 
the  end  of  which  time  numerous  spore-bearing  bacilli  can  often 
be  observed  microscopically.  The  culture  is  then  kept  at  80°  C. 


FIG.  121.— Tetanus  bacilli,  showing  flagella. 
Stained  by  Rd.  Mnir's  method,     x  1000. 


for  from  three-quarters  to  one  hour,  with  the  view  of  killing  all 
organisms  except  those  which  have  spored.  A  loopful  is  then 
added  to  glucose  gelatin,  and  roll-tube  cultures  are  made  in  the 
usual  way  and  kept  in  an  atmosphere  of  hydrogen  at  22°  C. ; 
after  five  days  the  plates  are  ready  for  examination.  Kitasato 
compares  the  colonies  in  gelatin  plates  to  those  of  the  b. 
subtilis.  They  consist  of  a  thick  centre  with  shoots  radiating 
out  on  all  sides.  They  liquefy  the  gelatin  more  slowly  than  the 
b.  subtilis.  This  method  of  isolation  is  not  always  successful, 
partly  because  along  with  the  tetanus  bacilli,  both  in  its  natural 


ISOLATION  OF  THE  BACILLUS 


419 


habitats  outside  the  body  and  in  the  pus  of  wounds,  other  spore- 
forming    obligatory   and 

facultative         anaerobes 

occur,  which  grow  faster 

than  the  tetanus  bacillus, 

and  thus  overgrow  it. 

(2)  If  in  any  discharge     / 

the  spore-bearing  tetanus    ,  ' 

bacilli  be  seen  on  micro-    * 

scopic  examination,  then 

a    method    of    isolation 

based  on  the  same  prin- 
ciple   as    the    last    may 

be    adopted.        Inocula-  ?"*"*  * 

tions  with  the  suspected 

material    are    made     in 

half  a  dozen  deep  tubes 

of   glucose   bouillon,  pre-       FIG.  122.—  Spiral  composed  of  numerous 

viously  raised   to  a  tem-         twisted  tiagella  of  the  tetanus  bacillus. 

perature  of  100°  C.  After      Staiue(l  b*  R(L  Muir's  raethod'     x  100°- 

inoculation      they      are 

again  placed  in  boiling  water  and  kept  for  varying  times,  say 

for    half    a    minute,  for 

one,  three,  four,  five,  and 

six  minutes  respectively. 

^  They   are   then   plunged 

-^—  '         in   cold  water   till  cool, 

and  thereafter  placed  in 

j.1,  ^      ' «.-1*  —  j_^__ 


\ 


Fi<;.  l'J:J.  —  Trtiinus  liacilli  ;  some  of  which 
possess  sjxnvs.  From  a  culture  in  glucose 
agar,  incubated  for  three  days  at  37°  C. 
See  also  Plate  IV.,  Fig.  20. 

Stained  with  carbol-fuchsin.      x  1000. 


Bulloch,    may  be   employed.       The 


the  incubator  at  37°  C., 
in  the  hope  that  in  one 
or  other  of  the  tubes  all 
the  organisms  present 
will  have  been  killed, 
except  the  tenanus  spores 
which  can  develop  in 
pure  culture.  A  series 
of  deep  glucose  agar 
tubes  may  also  be  in- 
oculated from  the  series 
of  bouillon  tubes. 

(3)    Some  method   of 
anaerobically        making 
plates,   such   as   that  of 
isolation    of    the    tetanus 


420 


TETANUS 


bacillus  is  in  many  cases  a  difficult  matter,  and  several  methods 
should  always  be  tried. 

Characters  of  Cultures. — Pure  cultures  having  been  obtained, 
sub-cultures  can  be  made  in  deep  upright  glucose  gelatin  or 
agar  tubes.  On  f/lucose  gelatin  in  such  a  tube  there  commences, 
an  inch  or  so  below  the  surface,  a  growth 
consisting  of  fine  straight  threads,  rather 
longer  in  the  lower  than  in  the  upper  parts 
of  the  tube,  radiating  out  from  the  needle 
track  (Fig.  124).  Slow  liquefaction  of  the 
gelatin  takes  place,  with  slight  gas  formation. 
In  agar  the  growth  is  somewhat  similar, 
consisting  of  small  nodules  along  the  needle 
track,  with  irregular  short  offshoots  passing 
out  into  the  medium  (Fig.  128,  A).  There  is 
slight  formation  of  gas,  but,  of  course,  no 
liquefaction.  On  anaerobic  agar  plates 
colonies  have  under  a  low  power  a  feathery 
outline  (Fig.  125).  Growth  also  occurs  in 
blood  serum  and  also  in  glucose  bouillon 
under  anaerobic  conditions.  The  latter  is  the 
medium  usually  employed  for  obtaining  the 
soluble  products  of  the  organism.  There  is 
in  it  at  first  a  slight  turbidity,  and  later  a 
thin  layer  of  a  powdery  deposit  on  the  walls 
of  the  vessel.  All  the  cultures  give  out  a 
peculiar  burnt  odour  of  rather  unpleasant 
character. 

Conditions  of  Growth,  etc. — The  b.  tetani 
grows  best  at  37°  C.  The  minimum  growth 
temperature  is  about  14°  C.,  and  below  22°  C. 
growth  takes  place  very  slowly.  Growth 
takes  place  in  the  absence  of  oxygen,  the 
organism  being  an  anaerobe.  Sporulation 
may  commence  at  the  end  of  twenty-four 
hours  in  cultures  grown  at  37°  C., — much 
later  at  lower  temperatures.  Like  other 
spores,  those  of  tetanus  are  extremely  resistant.  They  can 
usually  withstand  boiling  for  five  minutes,  and  can  be  kept 
in  a  dry  condition  for  many  months  without  being  killed  or 
losing  their  virulence.  They  have  also  high  powers  of  resistance 
to  antiseptics. 

Pathogenic  Effects. — The  proof  that  the  b.  tetani  is  the  cause 
of  tetanus  is  complete.     It  can  be  isolated  in  pure  culture,  and 


FIG.  124.— Stab  cul- 
ture of  the  tetanus 
bacillus  in  glucose 
gelatin,  showing 
the  lateral  shoots 
(Kitasato). 
Natural  size. 


PATHOGENIC  EFFECTS  421 

when  re-injected  in  pure  culture  it  reproduces  the  disease.  It 
may  be  impossible  to  isolate  it  from  some  cases  of  the  disease, 
but  the  cause  of  this  very  probably  is  the  small  numbers  in 
which  it  sometimes  occurs. 

(a)  The  Disease  as  arising  naturally. — The  disease  occurs 
naturally,  chiefly  in  horses  and  in  man.  Other  animals  may, 
however,  be  affected.  There  is  usually  some  wound,  often  of 
a  ragged  character,  which  has  either  been  made  by  an  object 
soiled  with  earth  or  dung,  or  which  has  become  contaminated 
with  these  substances.  There  is  often  a  purulent  or  foetid  dis- 
charge, though  this  may  be  absent.  In  tetanus  following 


* 


Kic.  125. — Colonies  of  the  tetanus  bacillus  on  anaerobic 
agar  plates,  seven  days  old.      x  50. 

clean  operation  wounds,  catgut  ligatures  may  be  the  source  of 
infection.  Microscopic  examination  of  sections  may  show  at  the 
edges  of  the  infected  wound  necrosed  tissue  in  which  the  tetanus 
bacilli  may  be  very  numerous.  If  a  scraping  from  the  wound 
be  examined  microscopically,  bacilli  resembling  the  tetanus  bacillus 
may  be  recognised.  If  these  have  spored,  there  can  be  practically 
no  doubt  as  to  their  identity,  as  the  drumstick  appearance  which 
the  terminal  spore  gives  to  the  bacillus  is  not  common  among 
other  bacilli.  Care  must  be  taken,  however,  to  distinguish  it  from 
other  thicker  bacilli  with  oval  spores  placed  at  a  short  distance 
from  their  extremities,  such  forms  being  common  in  earth, 
etc.,  and  also  met  with  in  contaminated  wounds.  It  is 
important  to  note  that  the  wound  through  which  infection  has 


m  TETANUS 

taken  place  may  be  very  small,  in  fact,  may  consist  of  a  mere 
abrasion.  In  some  cases,  especially  in  the  tropics,  it  may  be 
merely  the  bite  of  an  insect.  The  absence  of  a  definite  channel 
of  infection  has  given  rise  to  the  term  "idiopathic"  tetanus. 
There  is,  however,  practically  no  doubt  that  all  such  cases  are 
true  cases  of  tetanus,  and  that  in  all  of  them  the  cause  is 
the  b.  tetani.  The  latter  has  also  been  found  in  the  bronchial 
mucous  membrane  in  some  cases  of  the  so-called  rheumatic 
tetanus,  the  cause  of  which  is  usually  said  to  be  cold ;  infection 
of  the  intestinal  mucosa  may  also  occur. 

The  pathological  changes  found  post  mortem  are  not  striking. 
There  may  be  haemorrhages  in  the  muscles  which  have  been  the 
subject  of  the  spasms.  These  are  probably  due  to  mechanical 
causes.  Naturally  it  is  in  the  nervous  system  that  we  look  for 
the  most  important  lesions.  Here  there  is  ordinarily  a  general 
redness  of  the  grey  matter,  and  the  most  striking  feature  is  the 
occurrence  of  irregular  patches  of  slight  congestion  which  are  not 
limited  particularly  to  grey  or  white  matter,  or  to  any  tract  of 
the  latter.  These  patches  are  usually  best  marked  in  the  grey 
matter  of  the  medulla  and  pons.  Microscopically  there  is  little 
of  a  definite  nature  to  be  found.  There  is  congestion,  and  there 
may  be  minute  haemorrhages  in  the  areas  noted  by  the  naked 
eye.  The  ganglion  cells  may  show  appearances  which  have 
been  regarded  as  degenerative  in  nature,  and  similar  changes 
have  been  described  in  the  white  matter.  The  only  marked 
feature  is  thus  a  vascular  disturbance  in  the  central  nervous 
system,  with  a  possible  tendency  to  degeneration  in  its  specialised 
cells.  Both  of  these  conditions  are  probably,  due  to  the  action 
of  the  toxins  of  the  bacillus.  In  the  case  of  the  cellular  degenera- 
tions the  cells  have  been  observed  to  return  to  the  normal  under 
the  curative  influence  of  the  antitoxins  (vide  infra).  In  the 
other  organs  of  the  body  there  are  no  constant  changes. 

We  have  said  that  the  general  distribution  of  pathogenic 
bacteria  throughout  the  body  is  probably  a  relative  phenomenon, 
and  that  bacteria  usually  found  locally  may  occur  generally,  and 
vice  versa.  With  regard  to  the  tetanus  bacillus,  it  is,  however, 
probably  the  case  that  very  rarely,  if  ever,  are  the  organisms 
found  anywhere  except  in  the  local  lesion. 

(b)  The  Artificially-produced  Disease. — The  disease  can  be 
communicated  to  animals  by  any  of  the  usual  methods  of  inocula- 
tion, but  does  not  arise  in  animals  fed  with  bacilli,  whether 
these  contain  spores  or  not.  Kitasato  found  that  pure  cultures, 
injected  subcutaneously  or  intravenously,  caused  death  in  mice, 
rats,  guinea-pigs,  and  rabbits.  In  mice,  symptoms  appear  in  a 


TOXINS  OF  THE  TETANUS  BACILLUS         423 

day,  and  death  occurs  in  two  or  three  days,  after  inoculation 
with  a  loopful  of  a  bouillon  culture.  The  other  animals 
mentioned  require  larger  doses,  and  death  does  not  occur  so 
rapidly.  Usually  in  animals  injected  subcutaneously  the  spasms 
begin  in  the  limb  nearest  the  point  of  inoculation.  In  the 
case  of  intravenous  inoculation  the  spasms  begin  in  the 
extensor  muscles  of  the  trunk,  as  in  the  natural  disease  in  man. 
After  death  there  is  found  slight  hypenemia  without  pus  forma- 
tion, at  the  seat  of  inoculation.  The  bacilli  diminish  in  number, 
and  may  be  absent  at  the  time  of  death.  The  organs  generally 
slio\\  little  change. 

Kitasato  stated  that  in  his  earlier  experiments  the  quantity  of 
culture  medium  injected  along  with  the  bacilli  already  contained 
enough  of  the  poisonous  bodies  formed  by  the  bacilli  to  cause 
death.  The  symptoms  came  on  sooner  than  by  the  improved 
method  mentioned  below,  and  were,  therefore,  due  to  the  toxins 
already  present.  In  his  subsequent  work,  therefore,  he  employed 
splinters  of  wood  soaked  in  cultures  in  which  spores  were 
present,  and  subsequently  subjected  for  one  hour  to  a  tempera- 
ture of  80"  C.  The  latter  treatment  not  only  killed  all  the 
Yegetative  forms  of  the  organism,  but,  as  we  shall  see,  was 
sufficient  to  destroy  the  activity  of  the  toxins.  When  such 
splinters  are  introduced  subcutaneously,  death  results  by  the 
development  of  the  spores  which  they  carry.  In  this  way  he 
completed  the  proof  that  the  bacilli  by  themselves  can  form 
toxins  in  the  body  and  produce  the  disease.  Further,  if  a 
small  quantity  of  garden  earth  be  placed  under  the  skin  of  a 
mouse,  death  from  tetanus  takes  place  in  a  great  many  cases. 
[Sometimes,  however,  in  such  circumstances  death  occurs  with- 
out tetanic  symptoms,  and  is  not  due  to  the  tetanus  bacillus  but 
to  the  bacillus  of  malignant  cedema,  which  also  is  of  common 
occurrence  in  the  soil  (vide  infrd)J\  By  such  experiments, 
supplemented  by  the  culture  experiments  mentioned,  the  natural 
habitats  of  the  b.  tetani,  as  given  above,  have  become  known. 

The  Toxins  of  the  Tetanus  Bacillus. — The  tetanus  bacillus 
being  thus  accepted  as  the  cause  of  the  disease,  we  have  to 
consider  how  it  produces  its  pathogenic  effects. 

Almost  contemporaneously  with  the  work  on  diphtheria  was  the 
attempt  made  with  regard  to  tetanus  to  explain  the  general  symptoms 
by  supposing  that  the  bacillus  could  excrete  soluble  poisons.  The 
earlier  results,  in  which  certain  bases,  tetanin  and  tetanotoxin,  were 
said  to  have  been  isolated,  have  only  a  historic  interest,  as  they  were 
obtained  by  faulty  methods.  In  1890,  Brieger  and  Fraenkel  announced 
that  they  had  isolated  a  toxalbumin  from  tetanus  cultures,  and  this  body 
\\;is  independently  discovered  by  Faber  in  the  same  year.  Brieger  and 


424  TETANUS 

Fraenkel's  body  consisted  practically  of  an  alcoholic  precipitate  from 
filtered  cultures  in  bouillon,  and  was  undoubtedly  toxic.  Within  recent 
years  such  attempts  to  isolate  tetanus  toxins  in  a  pure  condition  have 
practically  been  abandoned,  and  attention  has  been  turned  to  the 
investigation  of  the  physiological  effects  either  of  the  crude  toxin 
present  in  filtered  bouillon  cultures,  or  of  the  precipitate  produced  from 
the  same  by  ammonium  sulphate  (cf.  p.  195). 

The  toxic  properties  of  bacterium-free  filtrates  of  pure 
cultures  of  the  b.  tetani  were  investigated  in  1891  by  Kitasato. 
This  observer  found  that  when  the  filtrate,  in  certain  doses,  was 
injected  subcutaneously  or  intravenously  into  mice,  tetanic  spasms 
developed,  first  in  muscles  contiguous  to  the  site  of  inoculation, 
and  later  all  over  the  body.  Death  resulted.  He  found  that 
guinea-pigs  were  more  susceptible  than  mice,  and  rabbits  less  so. 
In  order  that  a  strongly  toxic  bouillon  be  produced,  it  must 
originally  have  been  either  neutral  or  slightly  alkaline.  Kitasato 
further  found  that  the  toxin  was  easily  injured  by  heat.  Exposure 
for  a  few  minutes  at  65°  C.  destroyed  it.  It  was  also  destroyed 
by  twenty  minutes'  exposure  at  60°  C.,  and  by  one  and  a  half 
hours'  at  55°  C.  Drying  had  no  effect.  It  was,  however, 
destroyed  by  various  chemicals  such  as  pyrogallol  and  also  by 
sunlight. 

In  anaerobic  bouillon  cultures  the  maximum  toxicity  is  de- 
veloped in  from  ten  to  fifteen  days.  Behring  pointed  out  that 
after  the  filtration  of  cultures  containing  toxin,  the  latter  may 
very  rapidly  lose  its  power,  and  in  a  few  days  may  only  possess 
yj^th  of  its  original  toxicity.  This  he  attributed  to  such  factors 
as  temperature  and  light,  and  especially  to  the  action  of  oxygen. 
Toxins  should  thus  have  a  layer  of  toluol  floated  on  the  surface 
and  be  kept  in  a  cool,  dark  place.  The  effect  of  harmful  agents 
on  the  crude  toxin  is  apparently  to  cause  a  degeneration  of  the 
true  toxin  so  as  to  form  what  it  is  convenient  at  present  to  call 
toxoids  similar  to  those  produced  in  the  case  of  diphtheria  toxin, 
and  it  is  also  true  here  that  the  toxoids  while  losing  their 
toxicity  may  still  retain  their  power  of  producing  immunity 
against  the  potent  toxin.  Further,  altogether  apart  from  the 
occurrence  side  by  side  in  the  crude  toxin  of  strong  and  weak 
poisons,  it  has  been  shown  that  such  crude  toxin  contains  toxic 
substances  of  probably  quite  a  different  nature.  Ehrlich  has 
shown  that  besides  the  predominant  spasm-producing  toxin 
(called  by  him  tetanospasmin),  there  exists  in  crude  toxin  a 
poison  capable  of  producing  the  solution  of  certain  red  blood 
corpuscles.  This  hsemolytic  agent  he  calls  tetanolysin.  It  does 
not  occur  in  all  samples  of  crude  tetanus  toxin,  nor  is  it  found 


TOXINS  OF  THE  TETANUS  BACILLUS         425 

when  a  bouillon  culture  of  the  bacillus  is  filtered  through 
I>orcelain.  To  obtain  it  the  fresh  culture  must  be  treated  by 
ammonium  sulphate,  as  described  in  the  method  of  obtaining 
concentrated  toxins  (p.  195).  This  substance  also  has  the 
power  of  originating  an  antitoxin,  so  that  certain  antitetanic  sera 
can  protect  red  blood  corpuscles  against  its  action.  Madsen, 
studying  the  interactions  of  this  anti-tetanolysin  with  the 
tetanolysin,  has  shown  that  phenomena  can  be  demonstrated 
similar  to  those  noted  by  Ehrlich  as  occurring  with  diphtheria 
toxin,  and  which  the  latter  interpreted  as  indicating  the  presence 
of  degenerated  toxins  (toxoids)  in  the  crude  poison.  With 
tetanus  as  with  diphtheria  toxin  the  action  of  an  acid  is  to 
cause  an  apparent  disappearance  of  toxicity,  but  if  before  a 
certain  time  has  elapsed  the  acid  be  neutralised  by  alkali,  then  a 
degree  of  the  toxicity  returns. 

As  with  other  members  of  the  group,  nothing  is  known  of  the 
nature  of  tetanus  toxin.  Uschinsky  has  found  that  the  tetanus 
bacillus  can  produce  its  toxin  when  growing  in  a  fluid  containing 
no  proteid  matter.  The  toxin  may  thus  be  formed  independently 
of  the  breaking  up  of  the  proteins  on  which  the  bacillus  may  be 
living,  though  the  latter  no  doubt  has  a  digestive  action  on  such 
a  protein  as  gelatin.  There  is,  however,  evidence  that  peptic 
digestion  and  toxin  formation  are  due  to  different  vital  processes 
on  the  part  of  the  tetanus  bacillus. 

Whatever  the  nature  of  the  toxin  is,  it  is  undoubtedly  one 
of  the  most  powerful  poisons  known.  Even  with  a  probably 
impure  toxalbumin  Brieger  found  that  the  fatal  dose  for  a 
mouse  was  '0005  of  a  milligramme.  If  the  susceptibility  of 
man  be  the  same  as  that  of  a  mouse,  the  fatal  .dose  for  an  average 
adult  would  have  been  '23  of  a  milligramme,  or  about  -^V^ths 
of  a  grain.  Animals  differ  very  much  in  their  susceptibilities 
to  the  action  of  tetanus  toxin.  According  to  v.  Lingelsheim,  if 
the  minimal  lethal  dose  per  gramme  weight  for  a  horse  be  taken 
as  unity,  that  for  the  guinea-pig  would  be  6  times  the  amount, 
the  mouse  12,  the  goat  24,  the  dog  about  500,  the  rabbit  1800, 
the  cat  6000,  the  goose  12,000,  the  pigeon  48,000,  and  the 
hen  360,000. 

A  striking  feature  of  the  action  of  tetanus  toxin  is  the 
occurrence  of  a  definite  incubation  period  between  the  introduc- 
tion of  the  toxin  into  an  animal's  body  and  the  appearance  of 
symptoms.  The  incubation  period  varies  according  to  the  species 
of  animal  employed,  and  the  path  of  infection.  In  the  guinea- 
pi  i:  it  is  from  thirteen  to  eighteen  hours,  in  the  horse  five  days, 
and  the  incubation  is  shorter  when  the  poison  is  introduced  into 


426  TETANUS 

a  vein  than  when  injected  subcutaneously.  In  man  the  period 
between  the  receiving  of  an  injury  and  the  appearance  of  tetanic 
symptoms  is  from  two  to  fourteen  days. 

With  regard  to  the  action  of  the  toxin,  it  has  been  shown  to 
have  no  effect  on  the  sensory  or  motor  endings  of  the  nerves. 
It  acts  solely  as  an  exciter  of  the  reflex  excitability  of  the  motor 
cells  in  the  spinal  cord.  The  motor  cells  in  the  pons  and 
medulla  are  also  affected,  and  to  a  much  greater  degree  than 
those  in  the  cerebral  cortex.  When  injected  subcutaneoitsly 
the  toxin  is  absorbed  into  the  nerves,  and  thence  finds  its  way 
to  that  part  of  the  spinal  cord  from  which  these  nerves  spring. 
This  explains  the  fact  that  in  some  animals  the  tetanic  spasms 
appear  first  in  the  muscles  of  the  part  in  which  the  inoculation 
has  taken  place.  This  is  not  the  case  with  man,  in  whom  usually 
the  first  symptoms  appear  in  the  neck.  After  subcutaneous 
injection  of  toxin,  part  finds  its  way  into  the  blood  stream,  and 
if  infected  animals  be  killed  during  the  incubation  period  there 
is  often  evidence  of  toxin  in  the  blood  and  solid  organs.  In  the 
guinea-pig  there  is  little  doubt  that  tetanus  toxin  has  an  affinity 
solely  for  the  nervous  system.  In  other  animals,  e.(j.  the 
rabbit,  an  affinity  may  exist  in  other  organs,  and  the  fixation  of 
the  poison  in  such  situations  may  give  rise  to  no  recognisable 
symptoms.  In  such  an  animal  as  the  alligator,  it  is  possible 
that  while  some  of  its  organs  have  an  affinity  for  tetanus  toxin 
its  nervous  system  has  none.  These  facts  are  of  great  scientific 
interest,  and  a  possible  explanation  of  them  will  be  discussed  in 
the  chapter  on  Immunity.  If  tetanus  toxin  be  introduced  into 
the  stomach  or  intestine,  it  is  not  absorbed,  but  to  a  large  extent 
passes  through  the  intestine  unchanged.  Evidence  that  any 
destruction  takes  place  is  wanting. 

Within  recent  years  some  important  light  has  been  shed  on 
the  mode  of  action  of  tetanus  toxin.  Marie  and  Morax  studied 
the  path  of  absorption  when  the  toxin  was  injected  into  the 
muscles  of  the  hind  limb.  The  sciatic  nerve  in  a  rabbit  was  cut 
near  the  spinal  cord  and  toxin  introduced  into  the  muscles  of  the 
same  side ;  after  some  hours  the  nerve  was  excised  and  introduced 
into  a  mouse — the  animal  died  of  tetanus.  But  if  the  nerve  were 
cut  near  the  muscles  and  the  same  procedure  adopted,  the  mouse 
did  not  contract  the  disease,  though  no  doubt  the  cut  nerve  had 
been  surrounded  by  lymph  containing  toxin.  If  the  same 
experiment  were  performed  and  an  excess  of  toxin  injected  into 
the  other  limb,  still  only  the  nerve  which  was  left  in  connection 
with  the  muscle  showed  evidence  of  the  presence  of  toxin.  From 
this  it  was  deduced  that  the  toxin  was  absorbed  by  the  end- 


TOXINS  OF  THE  TETANUS  BACILLUS        427 

plates  in  the  muscle  and  not  from  the  lymphatics  surrounding 
the  nerve.  It  was  further  shown  that  a  nerve  in  the  process  of 
degeneration  following  section  did  not  absorb  toxin  after  the 
manner  of  a  normal  nerve.  By  a  similar  method  it  was  shown 
that  the  absorption  by  the  nerve  was  fairly  rapid,  as  one  hour 
after  injection  the  toxin  was  present  in  it,  and  from  other 
experiments  the  view  was  put  forth  that  the  toxin  was  centripetal 
in  its  flow  and  did  not  pass  centrifugally  in  a  nerve  to  which  it 
artificially  gained  access.  Further  observations  have  been  made 
on  this  subject  by  Meyer  and  Ransom.  These  observers  found 
evidence  that  toxin  is  only  absorbed  by  the  motor  filaments  of  a 
nerve,  for  while  tetanus  could  be  produced  by  injection  into  a 
mixed  nerve  like  the  sciatic,  the  introduction  of  a  lethal  dose  into 
such  a  sensory  nerve  as  the  infra-orbital  was  not  followed  by 
disease  symptoms.  If  a  small  dose  of  toxin  be  injected  into  the 
sciatic  nerve,  it  reaches  the  corresponding  motor  cells  of  the  cord, 
and  a  local  tetanus  of  the  muscles  supplied  by  the  nerve  results. 
With  a  larger  dose  the  poison  passes  across  the  commissure  to 
the  corresponding  cells  of  the  other  side,  and  if  still  further 
excess  is  present  it  passes  up  the  cord  to  higher  centres.  The 
affection  of  such  higher  centres  can  be  prevented  by  section  of 
the  cord.  Meyer  and  Hansom  hold  that  when  toxin  is  injected 
subcutaneously  or  intravenously,  it  only  acts  by  being  absorbed 
by  the  end-plates  in  muscles  and  thence  passes  to  the  cord,  and 
they  consider  that  the  incubation  period  is  to  be  explained  by  the 
time  taken  for  this  extended  passage  to  occur.  In  this  connection 
they  point  out  that  it  is  in  the  larger  animals"  where  the  nerve 
path  is  longest,  that  the  incubation  period  is  also  long.  Like 
Marie  and  Morax,  they  believe  that  absorption  of  toxin  by 
its  bathing  the  lateral  aspects  of  uninjured  nervous  structures 
does  not  occur.  In  support  of  this  they  bring  forward  the 
observation  that  when  intravenous  injection  is  practised,  the 
occurrence  of  tetanus  in  a  part  of  the  body  can  be  precipitated 
by  the  injection  of  a  drop  of  normal  saline  into  the  correspond- 
ing part  of  the  cord, — sufficient  injury  being  thus  caused  to  allow 
the  toxin  in  the  surrounding  lymph  to  obtain  access  to  the 
nervous  elements.  With  regard  to  the  action  of  tetanus  toxin, 
Meyer  and  Ransom  believe  that  there  is  a  double  effect  on  the 
nerve  cells — first,  an  exaggeration  of  the  normal  tonus,  which 
accounts  for  the  continuous  stiffness  of  the  muscles,  and 
secondly,  an  increase  in  reflex  irritability,  which  is  a  pro- 
minent factor  in  the  recurring  spasms.  While  no  absorption 
of  toxin  takes  place  by  sensory  filaments,  they  have  found 
evidence  of  atfection  of  the  sensory  apparatus  in  the 


428  TETANUS 

occurrence  of  what  they  call  tetanus  [dolorosus.  This  is  a 
great  hypersesthesia  and  a  paroxysmal  hyperalgesia  which  can  be 
caused  by  injecting  toxin  into  the  spinal  cord  or  into  a  sensory 
root  on  the  spinal  side  of  the  posterior  root  ganglion.  These 
symptoms  are  unaccompanied  by  motor  spasms,  but  the  animal 
may  die  from  exhaustion.  The  same  observers  have  also  made 
interesting  observations  on  the  action  of  antitoxin.  They  found 
that  the  injection  of  this  substance  into  the  course  of  a  mixed 
nerve  could  prevent  toxin  from  passing  up  to  the  cord,  but  that  if 
antitoxin  were  injected  even  in  great  excess  intravenously,  and  a 
short  time  thereafter  toxin  were  introduced  into  a  nerve,  the 
death  of  the  animal  was  not  prevented.  This  they  attribute 
to  the  fact  that  antitoxin  can  only  neutralise  the  toxin  which 
is  still  circulating  in  the  blood.  This  is  a  very  far-reaching 
conclusion,  as  it  throws  doubt  on  what  has  been  held  to  be  a 
possibility,  namely,  that  toxin  can  be  actually  detached  from 
cells  in  which  it  is  already  anchored.  But  a  still  more 
significant  observation  was  made,  for  in  one  case  of  an  animal 
actively  immunised  against  tetanus,  and  which  contained  in  its 
serum  a  considerable  quantity  of  antitoxin,  the  injection  of  toxin 
into  the  sciatic  nerve  was  followed  by  tetanus.  This  would 
appear  to  militate  against  Ehrlich's  position  that  antitoxin  is 
manufactured  in  the  cells  which  are  sensitive  to  the  toxin  (see 
Immunity). 

Reference  may  here  be  made  to  the  effects  of  injecting  tetanus 
toxin  into  the  brain  itself,  as  investigated  by  Roux  and  Borrel. 
It  was  found  th'at  the  ordinary  type  of  the  disease  was  not 
produced,  but  what  these  observers  called  "cerebral  tetanus." 
This  consisted  of  general  unrest,  symptoms  of  a  psychic  character 
(apparent  hallucinations,  fear,  etc.),  and  epileptiform  convul- 
sions. Death  occurred  in  from  twelve  to  twenty  hours  without 
any  true  tetanic  spasms.  In  this  manifestation  of  tetanus  the 
incubation  period  was  much  shorter  than  with  subcutaneous 
injection,  and  the  fatal  dose  was  one  twenty-fifth  of  the  minimal 
subcutaneous  dose.  Further,  the  injection  of  antitoxin  forty-eight 
to  ninety-six  hours  previously  did  not  prevent  an  animal  from 
succumbing  to  the  intracerebral  inoculation.  In  the  light  of 
what  has  been  already  said,  these  results  would  seem  to  indicate 
a  special  effect  of  the  toxin  when  brought  into  direct  contact 
with  the  protoplasm  of  the  brain  cells. 

We  have  seen  that  unless  suitable  precautions  are  adopted  in 
experiments  with  tetanus  cultures  in  animals,  death  results  not 
from  the  multiplication  of  the  bacilli,  but  from  an  intoxication 
with  toxin  previously  existent  in  the  fluid  in  which  the 


IMMUNITY  AGAINST  TETANUS  429 

bacilli  have  been  growing.  According  to  Vaillard,  if  spores 
rendered  toxin-free,  by  being  kept  for  a  sufficient  time  at 
80°  C.,  are  injected  into  an  animal,  death  does  not  take 
place.  It  was  found,  however,  that  such  spores  can  be 
rendered  pathogenic  by  injecting  along  with  them  such  chemicals 
as  lactic  acid,  by  injuring  the  seat  of  inoculation  so  as  to  cause 
effusion  of  blood,  by  fracturing  an  adjacent  bone,  by  introducing 
a  mechanical  irritant  such  as  soil  or  a  splinter  of  wood  (as  in 
Kitasato's  experiments),  or  by  the  simultaneous  injection  of 
other  bacteria  such  as  the  staphylococcus  j)t/ogenes  aureus.  These 
facts,  especially  the  last,  throw  great  light  on  the  disease  as  it 
occurs  naturally,  for  tetanus  results  especially  from  wounds 
which  have  been  accidentally  subjected  to  conditions  such  as 
those  enumerated.  Kitasato  now  holds  that  in  the  natural 
infection  in  man,  along  with  tetanus  spores,  the  presence  of 
foreign  material  or  of  other  bacteria  is  necessary.  Spores  alone 
or  tetanus  bacilli  without  spores  die  in  the  tissues,  and  tetanus 
does  not  result. 

Immunity  against  Tetanus. — Antitetanic  Serum. — The  arti- 
ficial immunisation  of  animals  against  tetanus  has  received  much 
attention.  The  most  complete  study  of  the  question  is  found 
in  the  work  of  Hehring  and  Kitasato  in  Germany,  and  of  Tizzoni 
and  Cattani  in  Italy.  The  former  observers  found  that  such  an 
immunity  could  be  conferred  by  the  injection  of  very  small  and 
progressively  increasing  doses  of  the  tetanus  toxin.  The  degree 
of  immunity  attained,  however,  was  not  high.  Subsequent 
work  has  shown  that  the  less  rich  a  crude  toxin  is  in  modifica- 
tions of  the  true  toxin,  the  less  useful  it  is  for  immunisation 
procedures.  In  fact  it  is  doubtful  if  small  animals  can  be 
immunised  at  all  by  fresh  filtrates.  In  some  cases  it  has  been 
found  that  the  injection  of  non-lethal  doses  instead  of  commen- 
cing an  immunity  actually  increases  the  susceptibility  of  the 
animal.  This  observation  has  recently  acquired  fresh  interest 
from  its  falling  into  line  with  the  work  on  the  development 
of  supersensitiveness  to  proteids,  which  is  a  very  common 
phenomenon  (see  "  anaphylaxis "  under  Immunity).  More 
successful  in  producing  immunity  are  the  methods  of  accompany- 
ing the  early  injections  of  crude  toxin  with  the  subcutaneous 
introduction  of  small  doses  of  iodine  terchloride,  or  of  using 
toxin  which  has  been  acted  on  with  iodine  terchloride  or  with 
iodine  itself.  Tizzoni  and  Cattani  also  used  the  method  of 
administering  progressively  increasing  doses  of  living  cultures 
attenuated  in  various  ways,  e.y.  by  heat.  By  any  of  these  methods 
susceptible  animals  can  be  made  to  acquire  great  immunity,  not 


430  TETANUS 

only  against  many  times  the  fatal  dose  of  tetanus  toxin,  but 
also  against  injections  of  the  living  bacilli.  The  degree  of 
immunisation  thus  acquired  remains  in  existence  for  a  very  long 
time.  Not  only  so,  but  when  a  high  degree  of  immunity  has 
been  produced  by  prolonged  treatment,  it  is  found  that  the 
serum  of  immune  animals  possesses  the  capacity,  when  injected 
into  animals  susceptible  to  the  disease,  of  protecting  them 
against  a  subsequent  infection  with  a  fatal  dose  of  tetanus 
bacilli  or  toxin.  Further,  if  injected  subsequently  to  such 
infection,  the  serum  can  in  certain  cases  prevent  a  fatal  result, 
even  when  symptoms  have  begun  to  appear.  The  degree  of 
success  attained  depends,  however,  on  the  shortness  of  the  time 
which  has  elapsed  between  the  infection  with  the  bacilli  or  toxin 
and  the  injection  of  the  serum.  In  animals  where  symptoms 
have  fully  manifested  themselves  only  a  small  proportion  of 
cases  can  be  saved.  As  with  other  antitoxins,  there  is  no 
evidence  that  the  antitetanic  serum  has  any  detrimental  effect 
on  the  bacilli.  It  only  neutralises  the  effects  of  the  toxin. 
The  standardisation  of  the  antitetanic  serum  is  of  the  highest 
importance.  Behring  recommends  that  for  protecting  animals 
a  serum  should  be  obtained  of  which  one  gramme  will  protect 
1,000,000  grammes  weight  of  mice  against  the  minimum  fatal 
dose  of  the  bacillus  or  toxin.  A  mouse  weighing  twenty 
grammes  would  thus  require  "00002  gramme  of  the  serum  to 
protect  it  against  the  minimum  lethal  dose.  In  the  injection 
of  such  a  serum  subsequent  to  infection,  if  symptoms  have 
begun  to  appear,  1000  times  this  dose  would  be  necessary;  a 
few  hours  later  10,000  times,  and  so  on. 

As  the  result  of  his  experiments,  Behring  aimed  at  obtaining 
a  curative  effect  in  the  natural  disease  occurring  in  man.  For 
this  purpose,  as  for  his  later  laboratory  experiments,  he  obtained 
serum  by  the  immunisation  of  such  large  animals  as  the  horse, 
the  sheep,  and  the  goat,  by  the  injection  of  toxin  accompanied 
at  first  with  the  injection  of  iodine  terchloride.  It  was  found 
that  the  greater  the  degree  of  the  natural  susceptibility  of  an 
animal  to  tetanus,  the  easier  was  it  to  obtain  a  serum  of  a  high 
antitetanic  potency.  The  horse  was,  therefore,  the  most 
suitable  animal.  If  now  we  take  for  granted  that  the  relative 
susceptibilities  of  man  and  the  mouse  towards  tetanus  are  nearly 
equal,  a  man  weighing  100  kilogrm.  would  require  '1  grm.  of 
the  serum  mentioned  above  to  protect  him  from  inoculation 
with  the  minimum  lethal  dose  of  bacilli  or  toxin.  If  symptoms 
had  begun  to  appear,  100  c.c.  at  once  would  be  necessary,  and 
as  the  injection  of  such  a  quantity  might  be  inconvenient, 


IMMUNITY  AGAINST  TETANUS  431 

Behring  recommended  that  for  man  a  more  powerful  serum 
should  be  obtained,  namely,  a  serum  of  which  one  gramme  would 
protect  100,000,000  grammes  weight  of  mice.1  The  potency  is 
maintained  for  several  months  if  precautions  are  taken  to 
avoid  putrefaction,  exposure  to  bright  light,  etc.  To  this  end 
•5  per  cent,  carbolic  acid  is  usually  added,  and  the  serum  is 
kept  in  the  dark.  In  a  case  of  tetanus  in  man,  100  c.c.  of 
such  a  serum  should  be  injected  within  twenty-four  hours  in 
five  doses,  each  at  a  different  part  of  the  body,  and  this 
followed  up  by  further  injections  if  no  improvement  takes 
place.  Intravenous  injection  of  the  antitoxin  has  also  been 
practised,  and,  in  cases  which  we  have  seen  treated  in  this  way, 
has  seemed  to  give  better  results  than  those  obtained  by  the 
subcutaneous  method.  The  serum  is  warmed  to  the  body 
temperature  and  slowly  introduced  into  a  vein  in  the  arm,  the 
pulse  and  respiration  being  carefully  watched  during  the 
proceeding.  Ten  to  twenty  c.c.  can  be  injected  every  few  hours, 
and  in  all  100  c.c.  should  be  given  in  as  short  a  time  as  possible. 
Henderson  Smith  has  shown  that  when  antitoxins  to  toxins  of 
the  tetanus  group  are  injected  intravenously  a  high  concentration 
in  the  body  fluid  is  maintained  for  some  time,  and  the  op- 
portunity for  neutralisation  of  toxin  is  thus  great.  He  suggests 
that  both  intravenous  and  subcutaneous  injections  should  be 
simultaneously  practised.  The  former  gives  quickly  the  con- 
centration which  is  desirable,  and  when  the  antitoxin  injected 
intravenously  is  beginning  to  be  eliminated,  that  introduced 
hypodermically  comes  into  the  circulation  and  the  concentration 
is  maintained.  The  antitoxin  has  also  been  introduced  intra- 
cerebrally,  very  slow  injection  into  the  brain  substance  being 
practised,  but  no  better  results  have  been  obtained  than  by  the 
subcutaneous  method. 

Many  cases  of  human  tetanus  have  been  thus  treated,  but 
the  improvement  in  the  death-rate  has  not  been  nearly  so 
marked  as  that  which  has  occurred  in  diphtheria  under  similar 
circumstances.  As  in  the  case  of  diphtheria,  however,  the 
results  would  probably  be  better  if  more  attention  were  paid 
to  the  dosage  of  the  serum.  The  great  difficulty  is  that 
usually  we  have  not  the  opportunity  of  recognising  the 
presence  of  the  tetanus  bacilli  till  they  have  begun  to  manifest 
their  gravest  effects.  In  diphtheria  we  have  a  well-marked 
clinical  feature, — sore  throat, — which  draws  attention  to  the  pro- 

1  The  antitetauic  serum  sent  out  by  the  Pasteur  Institute  in  Paris  has  a 
strength  of  1  :  1,000,000,000.  Of  this  it  is  recommended  that  50  to  100  c.c. 
should  be  injected  subcutaneously  in  one  or  two  doses. 


432  TETANUS 

bable  presence  of  the  bacilli,  and  the  curative  agent  can  thus 
be  early  applied.  In  tetanus  the  wound  in  which  the  bacilli 
exist  may  be,  as  we  have  seen,  of  the  most  trifling  character, 
and  even  when  a  well-marked  wound  exists,  the  search  for  the 
bacilli  may  be  a  matter  of  difficulty.  Still  it  might  be  well, 
when  practicable,  that  every  ragged,  unhealthy-looking  wound, 
especially  when  contaminated  with  soil,  should,  as  a  matter  of 
routine,  be  examined  bacteriologically.  In  such  cases,  un- 
doubtedly, from  time  to  time  the  tetanus  bacillus  would  be  early 
detected,  and  treatment  could  be  undertaken  with  more  hope  of 
success  than  at  present.  However,  in  the  existing  state  of 
matters,  whenever  the  first  symptoms  of  tetanus  appear,  large 
doses,  such  as  those  above  indicated,  of  a  serum  whose  strength 
is  known,  should  be  at  once  administered.  In  giving  a  prognosis 
as  to  the  probable  result,  the  two  clinical  observations  on  which 
chief  reliance  ought  to  be  placed  are  the  presence  or  absence  of 
interference  with  respiration,  and  the  rapidity  with  which  the 
groups  of  muscles  usually  affected  are  attacked.  If  dyspnoea 
or  irregularity  in  respiration  or  rise  of  temperature  comes  on 
soon,  and  if  group  after  group  of  muscles  is  quickly  involved, 
then  the  outlook  is  extremely  grave.  In  addition  to  these 
points,  the  duration  of  the  incubation  period  is  of  high  im- 
portance in  forming  a  prognosis.  The  shorter  the  time  between 
the  infliction  of  a  wound  and  the  appearance  of  symptoms  the 
graver  is  the  outlook. 

The  theory  as  to  the  nature  of  antitoxic  action  will  be 
discussed  later  in  the  chapter  on  Immunity. 

Methods  of  Examination  in  a  case  of  Tetanus. — The 
routine  bacteriological  procedure  in  a  case  presenting  the 
clinical  features  of  tetanus  ought  to  be  as  follows  : — 

(a)  Microscopic. — Though  tetanus  is  not  a  disease  in  which 
the  discovery  of  the  bacilli  is  easy,  still  microscopic  examination 
should  be  undertaken  in  every  case.  From  every  wound  or 
abrasion  from  which  sufficient  discharge  can  be  obtained,  film 
preparations  ought  to  be  made  and  stained  with  any  of  the 
ordinary  combinations,  e.y.  carbol-fuchsin  diluted  with  five  parts 
of  water.  Drumstick-shaped  spore-bearing  bacilli  are  to  be 
looked  for.  The  presence  of  such,  having  characters  corre- 
sponding to  those  of  the  tetanus  bacilli,  though  not  absolutely 
conclusive  proof  of  identification,  is  yet  sufficient  for  all 
practical  purposes.  If  only  bacilli  without  spores  resembling 
the  tetanus  bacilli  are  seen,  then  the  identification  can  only  be 
provisional. 

The  microscopic  examination  of  wounds  contaminated  by  soil, 


MALIGNANT  OEDEMA  433 

etc.,  may,  as  we  have  said,  in  some  cases  lead  to  the  anticipation 
that  tetanus  will  probably  result. 

(b)  Cultivation. — The  methods  to  be  employed  in  isolating 
the  tetanus   bacilli  have  already  been  described  (p.  417).     It 
may    be   added,   however,  that   if  the  characteristic  forms  are 
not  seen  on  microscopic  examination  of  the  material  from  the 
wound,  they  may  often  be  found  by  inoculating  a  deep  tube  of 
one  of  the  glucose  media  with  such  material,  and  incubating  for 
forty-eight  hours  at  37°  C.     At  the  end  of  this  period,  spore- 
bearing  tetanus  bacilli  may  be  detected  microscopically,  though 
of  course  mixed  with  other  organisms. 

(c)  Inoculation. — Mice  and  guinea-pigs  are  the  most  suitable 
animals.     Inoculation  with  the  material  from  a  wound  should 
be  made  subcutaneously.     A  loopful  of  the  discharge  introduced 
at  the  root  of  the  tail  in  a  mouse  will  soon  give  rise  to  the 
characteristic   symptoms,   if  tetanus  bacilli  are  present.     With 
suspicious  organisms  isolated  by  culture  it  is  well  to  use  the 
splinter  method  (p.  423),  as  some  strains  of  the  b.  tetani  tend  to 
produce  little  toxin  in  artificial  media,    and  may  be  injected 
without  causing  tetanic  symptoms. 

MALIGNANT  (EDEMA  (Septicemie  de  Pasteur). 

The  organism  now  usually  known  as  the  bacillus  of  malignant 
cedema  is  the  same  as  that  first  discovered  by  Pasteur  and 
named  vibrion  septique.  He  described  its  characters,  distin- 
guishing it  from  the  anthrax  bacillus,  which  it  somewhat 
resembles  morphologically,  and  also  the  lesions  produced  by  it. 
He  found  that  it  grew  only  in  anaerobic  conditions,  but  was 
able  to  cultivate  it  merely  in  an  impure  state.  It  was  more 
fully  studied  by  Koch,  who  called  it  the  bacillus  of  malignant 
oedema,  and  pointed  out  that  the  disease  produced  by  it  is  not 
really  of  the  nature  of  a  septicsjemia,  as  immediately  after  death 
the  blood  is  practically  free  from  the  bacilli. 

"  Malignant  oedema "  in  the  human  subject  is  usually 
described  as  a  spreading  inflammatory  oedema  attended  with 
emphysema,  and  ultimately  followed  by  gangrene  of  the  skin 
and  subjacent  parts.  In  many  cases  of  this  nature  the  bacillus 
of  malignant  oedema  is  present,  associated  with  other  organisms 
which  aid  its  spread,  whilst  in  others  it  may  be  absent.  One  of 
us  has,  however,  observed  a  case  in  which  the  bacillus  was 
present  in  pure  condition.  Here  there  occurred  intense  oedema 
with  swelling  and  induration  of  the  tissues,  and  the  formation 
of  vesicles  on  the  skin.  Those  changes  were  attended  with  a 
28 


434 


MALIGNANT  (EDEMA 


reddish  discoloration  afterwards  becoming  livid.  Emphysema 
was  not  recognisable  until  the  limb  was  incised,  when  it  was 
detected,  though  in  small  degree.  Further,  the  tissues  had  a 
peculiar  heavy,  but  not  putrid,  odour.  The  bacillus,  which  was 
obtained  in  pure  culture,  was  present  in  enormous  numbers  in 
the  affected  tissues,  attended  by  cellular  necrosis,  serous 
exudation,  and  at  places  much  leucocytic  emigration.  The 


\ 


FIG.  126. — Film  preparation  from  the  affected  tissues  in  a  case  of 
malignant  oedema  in  the  human  subject,  showing  the  spore-bearing 
bacilli. 

Gentian-violet,      x  1000. 


picture,  in  short,  corresponded  with  that  seen  on  inoculating  a 
guinea-pig  with  a  pure  culture.  The  term  "  malignant  oedema  " 
should  be  limited  in  its  application  to  cases  in  which  the 
bacillus  in  question  is  present.  In  most  of  these  there  is  a 
mixed  infection  ;  in  some  the  bacillus  may  be  present  alone. 

This  organism-  has  a  very  widespread  distribution  in  nature, 
being  present  in  garden  soil,  dung,  and  various  putrefying 
animal  fluids ;  and  it  is  by  contamination  of  lacerated  wounds 
by  such  substances  that  the  disease  is  usually  set  up  in  the 


MICROSCOPICAL  CHARACTER  OF  CULTURES     435 


human  subject.  Malignant  oedema  can  be  readily  produced  by 
inoculating  susceptible  animals,  such  as  guinea-pigs,  with  garden 
soil.  The  bacillus  is  also  often  present  in  the  intestine  of  man 
and  animals,  and  has  been  described  as  occurring  in  some 
gangrenous  conditions  originating  in  connection  with  the 
intestine  in  the  human  subject. 

Microscopical  Characters. — The  bacillus  of  malignant  oedema 
is  a  comparatively  large  organism,  being  slightly  less  than  1  /A 
in  thickness,  that  is,  thinner  than  the  anthrax  bacillus.  It 
occurs  in  the  form  of  single  rods  3  to  10  /A  in  length,  but  both 
in  the  tissues  and  in 
cultures  in  fluids  it  fre- 
quently grows  out  into 
long  filaments,  which  may 
be  uniform  throughout  or 
segmented  at  irregular 
intervals.  In  cultures  on 
solid  media  it  chiefly 
occurs  in  the  form  of 
shorter  rods  with  some- 
what rounded  ends.  The 
rods  are  motile,  possessing 
several  laterally  placed 
flagella,  but  in  a  given 
si>ecimen,  as  a  rule,  only 
a  few  bacilli  show  active 
movement.  Under  suit- 
able conditions  they  form 
spores,  which  are  usually 
near  the  centre  of  the  rods 
and  have  an  oval  shape, 

their  thickness  somewhat  exceeding  that  of  the  bacillus  (Figs. 
126,  127).  The  bacillus  can  be  readily  stained  by  any  of  the 
basic  aniline  stains,  but  loses  the  colour  in  Gram's  method,  in 
this  way  differing  from  the  anthrax  bacillus. 

Characters  of  Cultures. — This  organism  grows  readily  at 
ordinary  temperature,  but  only  under  anaeroltic  conditions.  In 
a  puncture  culture  in  a  deep  tube  of  glucose  gelatin,  the  growth 
appears  as  a  whitish  line  giving  off  minute  short  processes,  the 
growth,  of  course,  not  reaching  the  surface  of  the  medium. 
Soon  lii|iiet'a«-tion  occurs  and  a  long  fluid  funnel  is  formed,  with 
turbid  contents  and  flocculent  masses  of  growth  at  the  bottom. 
At  the  same  time  bubbles  of  gas  are  given  off,  which  may  split 
up  tin-  gelatin.  The  colonies  in  gelatin  plates  under  anaerobic 


FIG.  127. — Bacillus  of  malignant  oedema, 
showing  spores.  From  a  culture  in 
glucose  agar,  incubated  for  tliree  days 
at  37°  C. 

Stained  with  weak  rarbol-fuchsin.     x  1000. 


436 


MALIGNANT  (EDEMA 


conditions  appear  first  as  small  whitish  points  which  under  the 
microscope  show  a  radiating  appearance  at  the  periphery, 
resembling  the  colonies  of  the  bacillus  subtilis.  Soon,  however, 
liquefaction  occurs  around  the  colonies,  and  spheres  with  turbid 
contents  result ;  gas  is  developed  around  the  colonies. 

In  deep  tubes  of  glucose  agar  at  37°  C.  growth  is  extremely 


FIG.  128. — Stab  cultures  in  agar,  five  days'  growth  at  37°  C. 
Natural  size. 

A.  Tetanus  bacillus.     B.  Bacillus  of  malignant  oedema.    C.  Bacillus 
of  quarter-evil  (Rauschbrand). 

rapid.  Along  the  line  of  puncture,  growth  appears  as  a  some- 
what broad  white  line  with  short  lateral  projections  here  and 
there  (Fig.  128,  B).  Here  also  gas  may  be  formed,  but  this  is 
most  marked  in  a  shake  culture,  in  which  the  medium  becomes 
cracked  in  various  directions,  and  may  be  pushed  upwards  so 
high  as  to  displace  the  cotton-wool  plug.  The  cultures  possess 
a  peculiar  heavy,  though  not  putrid,  odour. 


EXPERIMENTAL  INOCULATION  437 

Spore  formation  occurs  above  20°  C.,  and  is  usually  well  seen 
within  forty-eight  hours  at  37°  C.  The  spores  have  the  usual 
high  powers  of  resistance,  and  may  be  kept  for  months  in  the 
dried  condition  without  being  killed. 

Experimental  Inoculation. — A  considerable  number  of  animals 
— the  guinea-pig,  rabbit,  sheep,  and  goat,  for  example — are 
susceptible  to  inoculation  with  this  organism.  The  ox  is  said 
to  be  quite  immune  to  experimental  inoculation,  though  it  can, 
under  certain  conditions,  contract  the  disease  by  natural  channels. 
The  guinea-pig  is  the  animal  most  convenient  for  experimental 
inoculation.  When  the  disease  is  set  up  in  the  guinea-pig  by 
subcutaneous  inoculation  with  garden  soil,  death  usually  occurs 
in  about  twenty-four  to  forty-eight  hours.  There  is  an  intense 
inflammatory  oedema  around  the  site  of  inoculation,  which 
extends  over  the  wall  of  the  abdomen  and  thorax.  The  skin 
and  subcutaneous  tissue  are  infiltrated  with  a  reddish-brown 
fluid  and  softened  ;  they  contain  bubbles  of  gas  and  are  at  places 
gangrenous.  The  superficial  muscles  are  also  involved.  These 
parts  have  a  very  putrid  odour.  The  internal  organs  are  con- 
gested, the  spleen  soft  but  not  much  enlarged.  In  such  condi- 
tions the  bacillus  of  malignant  oedema,  both  in  short  and  long 
forms,  will  be  found  in  the  affected  tissues  along  with  various 
other  organisms.  Spores  may  be  present,  especially  when  the 
examination  is  made  some  time  after  the  death  of  the  animal. 
If  the  animal  is  examined  immediately  after  death,  a  few  of  the 
bacilli  may  be  present  in  the  peritoneum  and  pleurae,  usually  in 
the  form  of  long  motile  filaments,  but  they  are  almost  invariably 
absent  from  the  blood.  A  short  time  after  death,  however,  they 
spread  directly  into  the  blood  and  various  organs,  and  may  then 
be  found  in  considerable  numbers. 

Subcutaneous  inoculation  with  pure  cultures  of  the  bacillus  of 
malignant  oedema  produces  chiefly  a  spreading  bloody  oedema, 
the  muscles  being  softened  and  partly  necrosed ;  but  there  is 
little  formation  of  gas,  and  the  putrid  odour  is  almost  absent. 

When  the  bacilli  are  injected  into  mice,  however,  they  enter 
and  multiply  in  the  blood  stream,  and  they  are  found  in  con- 
siderable numbers  in  the  various  organs,  so  that  a  condition  not 
unlike  that  of  anthrax  is  found.  The  spleen  also  is  much 
swollen. 

The  virulence  of  the  bacillus  of  malignant  oedema  varies  con- 
siderably in  different  cases,  and  it  always  becomes  diminished  in 
cultures  grown  for  some  time.  A  smaller  dose  produces  a 
fatal  result  when  injected  along  with  various  other  organisms 
(bacillus  prodigiosus,  etc.). 


438  BACILLUS  BOTtlLINUS 

Immunity. — Malignant  oedema  was  one  of  the  first  diseases 
against  which  immunity  was  produced  by  injections  of  toxins. 
The  filtered  cultures  of  the  bacillus  in  sufficient  doses  produce 
death  with  the  same  symptoms  as  those  caused  by  the  living 
organisms,  but  a  relatively  large  quantity  is  necessary.  Chamber- 
land  and  Koux  (1887)  found  that  if  guinea-pigs  were  injected 
with  several  non-fatal  doses  of  cultures  sterilised  by  heat  or  freed 
from  the  bacilli  by  filtration,  immunity  against  the  living 
organism  could  be  developed  in  a  comparatively  short  time. 
They  found  that  the  filtered  serum  of  animals  dead  of  the 
disease  is  more  highly  toxic,  and  also  gives  immunity  when 
injected  in  small  doses.  These  experiments  were  confirmed 
by  Sanfelice. 

Methods  of  Diagnosis. — In  any  case  of  supposed  malignant 
oedema,  the  fluid  from  the  affected  tissues  ought  first  to  be 
examined  microscopically,  to  ascertain  the  characters  of  the 
organisms  present.  Though  it  is  not  possible  to  identify  ab- 
solutely the  bacillus  of  malignant  oedema  without  cultivating  it, 
the  presence  of  spore-bearing  bacilli  with  the  characters  described 
above  is  highly  suspicious  (Fig.  126).  In  such  a  case  the  fluid 
containing  the  bacilli  should  be  first  exposed  to  a  temperature 
of  80°  C.  for  half  an  hour,  and  then  a  deep  glucose  agar  tube 
should  be  inoculated.  In  this  way  the  spore-free  organisms  are 
killed  off.  Pure  cultures  may  be  thus  obtained,  or  this  procedure 
may  require  to  be  followed  by  the  roll-tube  method  under 
anaerobic  conditions.  An  inoculation  experiment,  if  available, 
may  also  be  made  on  a  guinea-pig. 

BACILLUS  BOTULINUS. 

The  term  "  meat-poisoning  "  embraces  a  number  of  conditions 
produced  by  different  agents,  and  the  relation  of  the  bacillus  of 
Gaertner  to  one  class  of  case  has  already  been  discussed.  Another 
group  was  shown  by  van  Ermengem  in  1896  to  be  caused  by  an 
anaerobic  bacillus  to  which  he  gave  the  name  bacillus  botulinus. 
He  cultivated  the  organism  from  a  sample  of  ham,  the  inges- 
tion  of  which  in  the  raw  condition  had  produced  a  number  of 
cases  of  poisoning,  some  of  them  followed  by  fatal  result.  The 
symptoms  in  these  cases  closely  corresponded  with  those  occur- 
ring in  the  so-called  "  sausage  poisoning  "  met  with  from  time  to 
time  in  Germany  and  other  countries  where  sausages  and  ham 
are  eaten  in  an  imperfectly  cooked  condition.  Such  cases  form 
a  fairly  well-defined  group,  the  symptoms  in  which  are  chiefly 
referable  to  an  action  on  the  medulla,  and,  as  will  be  detailed 


MICROSCOPICAL  AND  CULTURAL  CHARACTERS    439 

below,  similar  symptoms  have  been  experimentally  produced 
by  means  of  the  bacillus  mentioned  or  its  toxins.  The  chief 
symptoms  of  this  variety  of  botulismus,  as  detailed  by  van 
Krmengem,  are  disordered  secretion  in  the  mouth  and  nose,  more 
or  less  marked  ophthalmoplegia,  externa  and  interna  (dilated 
pupil,  ptosis,  etc.),  dysphagia,  and  sometimes  aphagia  with 
aphonia,  marked  constipation  and  retention  of  urine,  and  in 
fatal  cases  interference  with  the  cardiac  and  respiratory  centres. 
Along  with  these  there  is  practically  no  fever  and  no  interference 
with  the  intellectual  faculties.  The  symptoms  commence  at 
earliest  twelve  to  twenty-four  hours  after  ingestion  of  the  poison. 
From  the  ham  in  question,  which  was  not  decomposed  in  the 
ordinary  sense,  van  Ermengem  obtained  numerous  colonies  of 
this  bacillus,  the  leading  characters  of  which  are  given  below. 
It  may  be  added  that  Romer  obtained  practically  the  same 
results  as  van  Ermengem  in  a  similar  condition,  and  that  the 
bacillus  botulinus  has  been  cultivated  by  Kempner  from  the 
intestine  of  the  pig. 

Microscopical  and  Cultural  Characters. — The  organism  is  a 
bacillus  of  considerable  size,  measuring  4  to  9  /x  in  length  and 
•9  to  1  '2  /x  in  thickness ;  it  has  somewhat  rounded  ends  and 
sometimes  is  seen  in  a  spindle  form.  It  is  often  arranged  in 
pairs,  sometimes  in  short  threads.  .  Under  certain  conditions 
it  forms  spores  which  are  oval  in  shape,  usually  terminal  in 
position,  and  a  little  thicker  than  the  bacilli.  It  is  a  motile 
organism  and  has  4  to  8  lateral  flagella  of  wavy  form.  It 
stains  readily  with  the  ordinary  dyes,  and  also  retains  the 
colour  in  Gram's  method,  though  care  must  be  employed  in 
decolorising. 

The  organisms  can  be  readily  cultivated  on  the  ordinary 
media,  but  only  under  strictly  anaerobic  conditions.  In  glucose 
gelatin  a  whitish  line  of  growth  forms  with  lateral  offshoots, 
but  liquefaction  with  abundant  gas  formation  soon  occurs.  In 
gelatin  plates  the  colonies  after  four  to  six  days  are  somewhat 
characteristic ;  they  appear  to  the  naked  eye  as  small  semi- 
transparent  spheres,  and  these  on  examination  under  a  low 
power  of  the  microscope  have  a  yellowish-brown  colour  and  are 
seen  to  be  composed  of  granules  which  show  a  streaming  move- 
ment, especially  at  the  periphery.  Cultures  in  glucose  agar 
resemble  those  of  certain  other  anaerobes;  there  is  abundant 
development  of  gas,  and  the  medium  is  split  up  in  various 
directions.  The  cultures  have  a  rancid,  though  not  foul,  odour, 
due  chiefly  to  the  development  of  butyric  acid.  The  optimum 
temperature  is  below  that  of  the  body,  namely,  between  20°  and 


440  BACILLUS  BOTULINUS 

30°  C.  ;  at  the    body  temperature  growth    is   slower   and  less 
abundant  and  spore  formation  does  not  occur. 

Pathogenic  Effects. — Like  the  tetanus  bacillus,  the  bacillus 
botulinus  has  little  power  of  nourishing  in  the  tissues,  whereas  it 
produces  a  very  powerful  toxin.  Van  Ermengem.  found  that  the 
characteristic  symptoms  could  be  produced  in  certain  animals 
by  administering  watery  extracts  of  the  infected  ham  or  cultures 
either  by  the  alimentary  canal  or  by  subcutaneous  injection. 
Here  also  there  is  a  period  of  incubation  of  not  less  than  six  to 
twelve  hours  before  the  symptoms  appear,  and  when  the  dose  is 
small  a  somewhat  chronic  condition  may  result,  in  which  local 
paralyses  form  a  striking  feature.  The  characteristic  effects 
can  also  be  produced  by  means  of  the  filtered  toxin  by  either  of 
the  methods  mentioned,  though  in  the  case  of  administration  by 
the  alimentary  canal  the  dose  requires  to  be  larger.  Here  also, 
as  in  the  case  of  the  tetanus  poison,  the  potency  of  the  toxin  is 
remarkable,  the  fatal  dose  for  a  guinea-pig  of  250  grm.  weight 
being  in  some  instances  '0005  c.c.  of  the  filtered  toxin.  In  cases 
of  poisoning  in  the  human  subject,  the  effects  would  accordingly 
appear  to  be  produced  by  absorption  of  the  toxin  from  the 
alimentary  canal ;  it  is  only  after  or  immediately  before  death 
that  a  few  bacilli  may  enter  the  tissues.  Van  Ermengem 
obtained  a  few  colonies  from  the  spleen  of  a  patient  who  had 
died  from  ham-poisoning.  The  properties  of  the  botulinus  toxin 
have  been  investigated,  and  have  been  found  to  correspond 
closely,  as  regards  relative  instability,  conditions  of  precipitation, 
combination  with  sensitive  cells  (i.e.  of  brain  and  cord),  etc., 
with  the  toxins  of  diphtheria  and  tetanus.  An  antitoxin  has 
also  been  prepared  by  Kempner  by  the  usual  methods,  and  has 
been  shown  not  only  to  have  a  neutralising  property,  but  to 
have  considerable  therapeutical  value  when  administered  some 
hours  after  the  toxin.  The  subject  has  been  recently  studied  by 
Leuchs,  and  he  has  found  that  the  combination  toxin-antitoxin 
can  be  split  up  by  the  action  of  acids  and  the  two  components 
recovered,  just  as  Morgenroth  showed  to  occur  in  the  case  of 
diphtheria  (p.  529).  The  direct  combining  affinity  of  the  toxin 
for  the  central  nervous  system  has  been  demonstrated  by  Kempner 
and  Schepilewsky  by  the  same  methods  as  Wassermann  and 
Takaki  employed  in  the  case  of  the  tetanus  toxin.  The  condition 
of  the  nerve  cells  in  experimental  poisoning  with  the  botulinus 
toxin  has  been  investigated  independently  by  Marinesco  and  by 
Kempner  and  Pollack,  and  these  observers  agree  as  to  the 
occurrence  of  marked  degenerative  changes,  especially  in  the 
motor  cells  in  tfre  spinal  cord  and  medulla,  Marinesco  also 


QUARTER-EVIL  441 

observed   hypertrophy  and  proliferation  of  the  neuroglia   cells 
around  them. 

These  observations,  therefore,  show  that  in  one  variety  of 
meat-poisoning  the  symptoms  are  produced  by  the  absorption  of 
the  toxins  of  the  bacillus  botulinus  from  the  alimentary  canal, 
and,  as  van  Ermengem  points  out,  it  is  of  special  importance  to 
note  that  the  meat  may  be  extensively  contaminated  with  this 
bacillus,  and  may  contain  relatively  large  quantities  of  its  toxins 
without  the  ordinary  signs  of  decomposition  being  present. 
The  production  of  an  extracellular  toxin  by  this  organism,  wdth 
extremely  potent  action  on  the  nervous  system,  is  a  fact  of  great 
scientific  interest,  and  has  a  bearing  on  the  etiology  of  other 
obscure  nervous  affections. 


QUARTER-EVIL  (GERMAN,  RAUSCHBRAND  ;  FRENCH,  CHARBON 
SYMPTOMATIQUE). 

The  characters  of  the  bacillus  need  be  only  briefly  described,  as,  so  far 
as  is  known,  it  never  infects  the  human  subject.  The  natural  disease, 
which  occurs  especially  in  certain  localities,  affects  chiefly  sheep,  cattle, 
and  goats.  Infection  takes  place  by  some  wound  of  the  surface,  and 
there  spreads  in  the  region  around,  inflammatory  swelling  attended  by 
bloody  u'denia  and  emphysema  of  the  tissues.  The  part  becomes  greatly 
swollen,  and  of  a  dark,  almost  black,  colour.  Hence  the  name  "black- 
leg" by  which  the  disease  is  sometimes  known.  The  bacillus  which 
produces  this  condition  is  present  in  large  numbers  in  the  affected  tissues, 
associated  with  other  organisms,  and  also  occurs  in  small  numbers  in  the 
blood  of  internal  organs.  For  the  isolation  of  the  bacillus,  Grassberger 
and  Schattenfroh  recommend  the  use  of  anaerobic  sugar  agar  plates  con- 
taining pieces  of  sterile  ox  flesh. 

The  bacillus  morphologically  closely  resembles  that  of  malignant 
fiidema.  Like  the  latter,  also,  it  is  a  strict  anaerobe,  and  its  conditions 
of  growth  as  regards  temperature  are  also  similar.  It  is,  however,  some- 
what thicker,  and  does  not  usually  form  such  long  filaments.  Moreover, 
the  spores,  which  are  of  oval  shape  and  broader  than  the  bacillus,  are 
almost  invariably  situated  close  to  one  extremity,  though  not  actually 
terminal  (Fig.  129).  The  characters  of  the  cultures,  also,  resemble 
those  of  the  bacillus  of  malignant  cedema,  but  in  a  stab  culture  in 
glucose  agar  there  are  more  numerous  and  longer  lateral  offshoots,  the 
growth  being  also  more  luxuriant  (Fig.  128,  c).  This  bacillus  is  actively 
motile,  and  possesses  numerous  lateral  flagella.  When  cultures  derived 
from  disease  conditions  are  continuously  subcultured  on  sugar  media,  they 
tend  to  lose  their  capacities  of  motility  and  spore  formation.  The 
organism  seems  to  occupy  a  position  somewhat  intermediate  between  the 
b.  saccharobutyricus  (v.  Klecki),  which  is  a  free  sugar  fermenter,  and  the 
b.  putrificus  (Bienstock),  which*has  great  powers  of  splitting  up  albumins. 

The  disease  can  be  readily  produced  in  various  animals,  e.g.  guinea- 
pigs,  by  inoculation  with  the  affected  tissues  of  diseased  animals,  and 
also  by  means  of  pure  cultures,  though  an  intramuscular  injection  of  a 
considerable  amount  of  the  latter  is  sometimes  necessary.  The  condition 
product  '1  in  this  way  closely  resembles  that  in  malignant  oedema,  though 


442 


QUARTER-EVIL 


there  is  said  to  be  more  formation  of  gas  in  the  tissues.  Rabbits  are 
more  resistant  to  this  disease,  whilst  they  are  comparatively  suscep- 
tible to  malignant  osdema.  As  in  the  case  of  tetanus,  inoculation  with 
living  spores  which  have  been  deprived  of  adherent  toxin  by  heat  does 
not  produce  the  disease.  A  toxin  can  be  separated  by  nitration  from 
cultures  of  bouillon  containing  5  per  cent,  glucose  and  a  thick  emulsion  of 
sterile  calcium  carbonate.  It  is  fairly  resistant  to  heat,  withstanding 
two  hours  at  70-75°  C.  without  being  destroyed,  and  it  is  also  very  rapid 
in  its  action,  being  capable  in  appropriate  dose  of  killing  a  horse  in  five 
minutes.  It  is  to  be  noted  as  an  important  fact,  that  while  fresHy 
isolated  cultures  possess  a  high  degree  of  virulence  they  may  have  little 

capacity  for  in  vitro  toxin 
production.  Grassberger  and 
Schattenfroh  state  that  there 
may  be  an  antagonism  be- 
tween maximum  virulence 
and  maximum  toxin  produc- 
tion. One  of  the  properties 
of  the  toxin  is  said  to  be  a 
capacity  for  killing  leuco- 
cytes. 

The  disease  is  one  against 
which  immunity  can  be  pro- 
duced in  various  ways,  and 
methods  of  preventive  inocu- 
lation have  been  adopted  in 
the  case  of  animals  liable  to 
suffer  from  it.  This  subject 
was  specially  worked  out 
by  Arloing,  Cornevin,  and 

Thomas,  and  later  by  others. 
FIG.  129.— Bacillus  ot  quarter-evil,  showing 

s^^r^  u<r  -• 

Stained  with  weak  carbol-fuchsiu.      xlOOO.    the.  intravenous    and    intra- 

peritoneal  routes)  with  a  non- 
fatal  dose  of  the  virus  (i.e. 

the  cedematous  fluid  found  in  the  tissues  of  affected  animals  and  which 
contains  the  bacilli),  or  by  injection  with  larger  quantities  of  the  virus 
attenuated  by  heat,  drying,  etc.  It  can  be  produced  also  by  cultures 
attenuated  by  heat  and  by  the  products  of  the  bacilli  obtained  by 
nitration  of  cultures.  An  antitoxin  has  been  produced  against  the  toxins 
of  the  bacillus,  and  a  method  of  protection  in  which  the  action  of  this 
antitoxin  is  combined  with  that  of  the  virus  has  been  used  (cf.  Anthrax, 
p.  348).  The  antitoxin  is  said  to  increase  the  chemiotactic  properties  of 
the  leucocytes. 

BACILLUS  AEROGENES  CAPSULATUS. 

This  bacillus,  though  sometimes  aiding  in  the  production  of  patho- 
•logical  changes,  is  chiefly  of  interest  on  account  of  the  extensive  gaseous 
development  to  which  it  gives  rise  in  the  tissues  post  mortem.  It  was 
described  by  Welch  and  Nuttall  in  1892  ;  it  is  now  recognised  as  being 
identical  with  an  organism  found  in  gaseous  phlegmon  by  E.  Fraenkel, 
and  called  by  him  the  bacillus  phlegmones  emphysematosce.  The  organism 
is  a  comparatively  large  one,  measuring  3  to  6  /x  in  length  and  having  a 


BACILLUS  AEROGENES  CAPSULATUS 


443 


thickness  about  the  same  as  that  of  the  anthrax  bacillus  ;  its  ends  are 
square  or  slightly  rounded  (Fig.  130).  It  often  occurs  in  pairs,  sometimes 
in  chains  ;  occasionally  filamentous  forms  are  met  with.  It  usually  shows 
a  well-marked  capsule,  hence  the  name  ;  it  is  non-motile  and  does  not 
form  spores.  It  stains  readily  with  the  basic  aniline  dyes  and  retains 
the  stain  in  Gram's  method.  It  grows  readily  on  the  ordinary  media, 
but  only  under  anaerobic  conditions  ;  the  optimum  temperature  is  that 
of  the  body,  growth  at  the  room  temperature  being  comparatively  slow. 
In  a  puncture  culture  in  agar  there  is  an  abundant  whitish  line  of  growth, 
wiih  somewhat  indented  margin  ;  the  individual  colonies  are  white  and 
of  rounded  or  oval  form.  There  is  practically  no  liquefaction  of  gelatin, 
though  this  medium  becomes  somewhat  softened  around  the  growth. 
In  all  cases  there  is  a  tendency  to  abundant  evolution  of  gas  in  the  cul- 
tures, and  this  is  especially 
marked  when  fermentable 
sugars  are  present. 

The  organism  appears  to 
be  the  most  frequent  cause 
of  rapid  gaseous  develop- 
ment in  the  blood  and 
organs  post-mortem,  this 
depending  upon  an  invasion 
of  the  blood  immediately 
before  death.  In  sucn 
cases,  even  within  twenty  - 
four  hours  under  ordinary 
conditions,  large  bubbles  of 
gas  may  be  present  in  the 
veins,  and  the  organs  may 
be  beset  with  gas-containing 
spheres  of  various  sizes  ;  the 
liver  is  usually  the  organ 
most  affected,  and  its  ap- 
pearance has  been  compared 
n  tliftf  nf  rrnv&rp  r-lfpp 
The  iuvasion^bTthis  ol^ 
ism  is  met  with  from  time 
to  time  in  puerperal  cases, 

an  1  also  in  connection  with  ulcerative  or  gangrenous  conditions  of  the 
intestine  ;  the  bacillus  is  also  found  not  infrequently  in  the  peritoneum 
in  cases  of  perforation.  Although  the  striking  changes  in  the  organs 
are  due  to  a  post-mortem  development  of  the  bacillus,  there  is  no 
doubt  that  its  entrance  into  the  blood  stream  often  hastens  death, 
and  may  in  some  instances  be  the  cause  of  it.  As  already  stated,  the 
organism  is  also  met  with  in  some  cases  of  spreading  oedema  with 
emphysema  as  a  leading  feature. 

When  tested  experimen  tally,  the  bacillus  by  itself  is  found  to  have 
little  pathogenic  action.  Injection  of  pure  cultures  in  rabbits  and 
guinea-pigs  may  be  followed  by  little  result,  but  sometimes  in  the  latter 
animals  "gaseous  phlegmon"  is  produced,  without  suppuration  unless 
other  organisms  are  present.  If  a  small  quantity  of  culture  be  injected 
intravenously,  c.y.  hi  a  rabbit,  and  then  the  animal  be  killed,  bubbles 
of  gas  are  rapidly  produced  in  the  blood  and  organs,  the  picture 
corresponding  with  that  in  the  human  cases, 


Fl£:    130.-Barillus    aerogenes    eapsulatus  ; 
nlm  preparation   from   bone-marrow  in  a 


444  FUSIFORM  ANAEROBIC  BACILLI 

FUSIFORM  ANAEROBIC  BACILLI  PATHOGENIC  TO  MAN. 

Babes  in  1884  described  organisms  of  this  type  in  a 
diphtheria-like  affection  of  the  fauces,  and  since  that  time  the 
presence  of  similar  organisms  has  been  noted  in  necrotic  inflam- 
mations, ulcerative  stomatitis,  noma,  and  like  affections.  The 
association  of  fusiform  bacilli  with  a  form  of  angina  has  been 
specially  recognised  since  the  work  of  Vincent  (1898-99);  and 
this  condition  often  goes  now  under  the  name  of  "Vincent's 
angina."  He  recognised  two  forms  of  the  affection — (a)  a 
diphtheroid  type  characterised  by  the  formation  of  a  firm 
yellowish-white  false  membrane,  very  like  that  of  diphtheria, 
associated  with  only  superficial  ulceration ;  and  (b)  an  ulcerative 
type  where  the  membrane  is  soft,  greyish,  and  foul-smelling, 
attended  with  ulceration  and  surrounding  oedema.  In  the 
former  type  fusiform  bacilli  are  present  alone;  in  the  latter, 
which  is  distinctly  the  commoner,  there  are  also  spirochsetes. 
The  fusiform  bacilli  are  thin  rods  measuring  on  the  average 
10  to  14  /x  in  length,  and  less  than  1  /x  in  thickness ;  they  are 
straight  or  slightly  curved  and  are  tapered  at  their  extremities. 
The  central  portion  often  stains  less  deeply  than  the  extremities, 
and  not  infrequently  shows  unstained  points  (Plate  I.,  Fig.  4). 
The  organisms  are  non-motile.  They  stain  fairly  deeply  with 
Loffler's  methylene-blue  or  with  weak  carbol-fuchsin.  They 
lose  the  stain  in  Gram's  method.  The  spirochsetes  are  long 
delicate  organisms  showing  several  irregular  curves,  and  are 
motile  ;  in  appearance  they  resemble  the  spirochsete  refringens 
and  similar  organisms  found  in  gangrenous  conditions.  They 
stain  less  deeply  than  the  bacilli.  Sometimes  they  are  numerous, 
sometimes  scanty ;  they  seem  to  be  similar  to  spirochsetse  found 
in  the  mouth  in  a  variety  of  other  conditions.  In  a  section 
through  the  false  membrane,  when  stained  with  methylene  or 
thionin  blue,  there  is  usually  to  be  seen  a  darkly  stained  band, 
a  short  distance  below  the  surface,  which  is  due  to  the  presence 
of  large  masses  of  the  fusiform  bacilli  closely  packed  together ; 
neither  they  nor  the  spirochsetes  appear  to  pass  deeply  into  the 
tissues.  Vincent's  results  have  been  confirmed  by  others,  and 
there  is  no  doubt  that  fusiform  bacilli,  of  which  there  are 
probably  several  species,  are  associated  with  various  spreading 
necrotic  conditions.  Cultures  of  fusiform  bacilli  have  been 
obtained  by  E  Hermann  and  by  Weaver  and  Tunnicliffe.  They 
grow  only  under  anaerobic  conditions,  and  the  best  media  are 
those  consisting  of  a  mixture  of  serum  or  blood  and  agar  (1  :  3). 
The  organisms  form  small  rounded  colonies  of  whitish  or 


FUSIFORM  ANAEROBIC  BACILLI  445 

yellowish  colour,  somewhat  like  those  of  a  streptococcus,  but 
rather  felted  in  appearance  on  the  surface.  Injections  of  pure 
cultures  in  animals  sometimes  produce  suppuration  but  never 
necrosis  (Ellermann).  Tunnicliffe  finds  that  the  spirochaetes 
are  only  stages  in  the  development  of  fusiform  bacilli,  as  cultures 
which  at  an  early  stage  show  only  fusiform  bacilli  afterwards 
contain  spirochsetes,  and  intermediate  forms  can  be  found.  These 
results,  however,  have  not  yet  been  confirmed.  It  is  also  to  be 
noted  that  fusiform  bacilli  are  sometimes  present  in  the  secretions 
of  the  mouth  in  normal  conditions,  and  may  occur  in  increased 
numbers  in  true  diphtheria. 


CHAPTER   XVIII. 

THE  CHOLERA  SPIRILLUM  AND  ALLIED 
ORGANISMS. 

Introductory. — It  is  no  exaggeration  of  the  facts  to  say  that 
previously  to  1883  practically  nothing  of  value  was  known 
regarding  the  nature  of  the  virus  of  cholera.  In  that  year 
Koch  was  sent  to  Egypt,  where  the  disease  had  broken  out,  in 
charge  of  a  Commission  for  the  purpose  of  investigating  its 
nature.  In  the  course  of  his  researches  he  discovered  the 
organism  now  generally  known  as  the  "  comma  bacillus "  or 
the  "cholera  spirillum."  He  also  obtained  pure  cultures  of 
the  organism  from  a  large  number  of  cases  of  cholera,  and 
described  their  characters.  The  results  of  his  researches  were 
given  at  the  first  Cholera  Conference  at  Berlin  in  1884. 

Since  Koch's  discovery,  and  especially  during  the  epidemic  in 
Europe  in  1892-93,  spirilla  have  been  cultivated  from  cases  of 
cholera  in  a  great  many  different  localities,  and  though  this 
extensive  investigation  has  revealed  the  invariable  presence  in 
true  cholera  of  organisms  resembling  more  or  less  closely  Koch's 
spirillum,  certain  difficulties  have  arisen.  For  it  has  been 
found  that  the  cultures  obtained  from  different  places  have 
shown  considerable  variations  in  their  characters,  and,  further, 
spirilla  which  closely  resemble  Koch's  cholera  spirillum  have 
been  cultivated  from  sources  other  than  cases  of  true  cholera. 
There  has  therefore  been  much  controversy,  on  the  one  hand, 
as  to  the  signification  of  these  variations, — whether  they  are 
to  be  regarded  as  indicating  distinct  species  or  merely  varieties 
of  the  same  species, — and,  on  the  other  hand,  as  to  the  means 
of  distinguishing  the  cholera  spirillum  from  other  species  which 
resemble  it.  These  questions  will  be  discussed  belo\v. 

In  considering  the  bacteriology  of  cholera,  it  is  to  be  borne  in 
mind  that  in  this  disease,  in  addition  to  the  evidence  of  great 
intestinal  irritation,  accompanied  by  profuse  watery  discharge, 
and  often  by  vomiting,  there  are  also  symptoms  of  general 

446 


MICROSCOPICAL  CHARACTERS  447 

systemic  disturbance  which  cannot  be  accounted  for  merely  by 
the  withdrawal  of  water  and  certain  substances  from  the 
system.  Such  symptoms  include  the  profound  general  prostra- 
tion, cramps  in  the  muscles,  extreme  cardiac  depression,  the 
cold  and  clammy  condition  of  the  surface,  the  subnormal 
temperature,  suppression  of  urine,  etc.  These,  taken  in  their 
entirety,  are  indications  of  a  general  poisoning  in  which  the 
circulatory  and  thermo  -  regulatory  mechanisms  are  specially 
involved.  In  some,  though  rare,  cases  known  as  cholera  sicca, 
general  collapse  occurs  with  remarkable  suddenness,  and  is 
rapidly  followed  by  a 
fatal  result,  whilst  there 
is  little  or  no  evacuation 
from  the  bowel,  though 
post-mortem  the  intestine 
is  distended  with  fluid 
contents.  As  the  char- 
acteristic  organisms  in 
cholera  are  found  only 
in  the  intestine,  the 
general  disturbances  are 
to  be  regarded  as  the 
result  of  toxic  substances 
absorbed  from  the  bowel. 
It  is  also  to  be  noted 
that  cholera  is  a  disease 

of  which  the  onset  and  Fl(J.  131. -Cholera  spirilla,  from  a  culture  on 
course  are  much  more  agar  of  twenty-four  hours'  growth, 

rapid  than  is  the  case  ill      Stained  with  weak  carbol-fuchsin.     xlOOO. 
most    infective    diseases, 

such  as  typhoid  and  diphtheria ;  and  also  that  recovery,  when 
it  takes  place,  does  so  more  quickly.  The  two  factors  to  be 
correlated  to  these  facts  are :  (a)  a  rapid  multiplication  of 
organisms,  (6)  the  production  of  rapidly  acting  toxins. 

The  Cholera  Spirillum. — Microscopical  Characters. — The 
cholera  spirilla,  as  found  in  the  intestines  in  cholera,  are  small 
organisms  measuring  about  1  *5  to  2  /A  in  length,  and  rather  less 
than  '5  in  thickness.  They  are  distinctly  curved  in  one  direction, 
hence  the  api>earance  of  a  comma  (Fig.  131);  most  occur 
singly,  but  some  are  attached  in  pairs  and  curved  in  opposite 
directions,  so  that  an  S-shai>e  results.  Longer  forms  are  rarely 
seen  in  the  intestine,  but  in  cultures  in  fluids,  as  is  especially 
well  seen  in  hanging-drop  preparations,  they  may  grow  into 
longer  spiral  filaments,  showing  a  large  number  of  turns.  In 


448 


CHOLERA 


: 


film  preparations  made  from  the  intestinal  contents  in  typical 

cases,  these  organisms  are 

<  "  *  present      in        enormous 

!.<?X  numbers   in   almost    pure 

VL  culture,     most      of      the 

^  ,        spirilla    lying  with  their 

-**     C.  long   axes    in    the    same 

direction,  so  as  to  give 
the  appearance  which 
Koch  compared  to  a  num- 
ber of  fish  in  a  stream. 

They  possess  very  active 
motility,  which  is  most 
marked  in  the  single 
forms.  When  stained  by 
the  suitable  methods  they 
are  seen  to  be  flagellated. 
Usually  a  single  terminal 
flagellum  is  present  at 
one  end  only  (Fig.  132). 
It  is  very  delicate,  and 
length  of  the  organism. 


FIG.  132. — Cholera  spirilla  stained  to  show 
the  terminal  flagella.  See  also  Plate 
IV.,  Fig  19.  xlOOO. 


the 


measures   four   or    five    times 
Cholera  spirilla   do  not 
form  spores.     In  old  cul- 
tures the  organisms  may 
present  great  variety  in 
size  and  shape.      Some 
are    irregularly    twisted 
filaments,  sometimes  glo-       ,  f 
bose,  sometimes  clubbed      ,  4j 
at  their  extremities,  and      * 
also    showing    irregular 
swellings     along      their 
course ;  others  are  short 
and     thick,     and     may 
have  the  appearance  of 
large  cocci,  often  stain- 
ing  faintly.     All    these 
changes    in    appearance 
are  to  be  classed  together 
as  involution  forms. 

Staining.  —  Cholera 
spirilla  stain  readily  with 
the  usual  basic  aniline  stains,  though  Loffler's  methylene-blue  or 


FIG.  133. — Cholera  spirilla  from  an  old  agar 
culture,  showing  irregularities  in  size  and 
shape,    with     numerous     faintly  -  stained 
coccoid  bodies — involution  forms. 
Stained  with  fuchsin.      x  1000. 


CULTIVATION  449 

weak  carbol-fuchsin  is  specially  suitable.  They  lose  the  stain 
in  Gram's  method. 

Distribution  within  the  Body. — The  chief  fact  in  this  con- 
nection is  that  the  spirilla  are  confined  to  the  intestine,  and  are 
not  present  in  the  blood  or  internal  organs.  In  cases  in  which 
there  is  the  characteristic  "  rice-water "  fluid  in  the  intestines, 
they  occur  in  enormous  numbers — almost  in  pure  culture.  The 
lower  half  of  the  small  intestine  is  the  part  most  affected.  Its 
surface  epithelium  becomes  shed  in  great  part,  and  the  flakes 
floating  in  the  fluid  consist  chiefly  of  masses  of  epithelial  cells 
and  mucus,  amongst  which  are  numerous  spirilla.  The  spirilla 
also  penetrate  the  follicles  of  Lieberkiilm,  and  may  be  seen 
lying  between  the  basement  membrane  and  the  epithelial  lining, 
which  becomes  loosened  by  their  action.  They  are,  however, 
rarely  found  in  the  connective  tissue  beneath,  and  never  pene- 
trate deeply.  Along  with  these  changes  there  is  congestion  of 
the  mucosa,  especially  around  the  Peyer's  patches  and  solitary 
glands,  which  are  somewhat  swollen  and  prominent.  In  some 
very  acute  cases  the  mucosa  may  show  general  acute  congestion, 
with  a  rosy  pink  colour  but  very  little  desquamation  of 
epithelium,  the  intestinal  contents  being  a  comparatively  clear 
fluid  containing  the  spirilla  in  large  numbers.  In  other  cases  of 
a  more  chronic  type,  the  intestine  may  show  more  extensive 
necrosis  of  the  mucosa  and  a  considerable  amount  of  haemorrhage 
into  its  substance,  along  with  formation  of  false  membrane  at 
places.  The  intestinal  contents  in  such  cases  are  blood-stained 
and  foul-smelling,  there  being  a  great  proportion  of  other 
organisms  present  besides  the  cholera  spirilla  (Koch). 

Cultivation. — (For  methods,  see  p.  459). 

The  cholera  spirillum  grows  readily  on  all  the  ordinary  media, 
and,  with  the  exception  of  that  on  potato,  growth  takes  place  at 
the  ordinary  room  temperature.  The  most  suitable  temperature, 
however,  is  that  of  the  body,  and  growth  usually  stops  about 
16°  C.,  though  in  some  cases  it  has  been  obtained  at  a  lower 
temperature.  Abundant  growth  occurs  on  media  with  suffi- 
ciently alkaline  reaction  to  inhibit  the  growth  of  many 
intestinal  bacteria,  e.g.  Dieudonne"'s  medium,  p.  44. 

Peptone  Gelatin. — On  this  medium  the  organism  grows  well 
and  produces  liquefaction.  In  puncture  cultivations  at  22°  C. 
a  whitish  line  appears  along  the  needle  track,  at  the  upper  part 
of  which  liquefaction  commences,  and  as  evaporation  quickly 
occurs,  a  small  bell-sliaprd  <lcpression  forms,  which  gives  the 
appearance  of  an  air-bubble.  On  the  fourth  or  fifth  day  we  get 
the  following  appearance:  there  is  at  the  surface  the  bubble- 
29 


450  CHOLERA 

shaped  depression  ;  below  this  there  is  a  funnel-shaped  area  of 
liquefaction,  the  fluid  being  only  slightly  turbid,  but  showing  at 
its  lower  end  thick  masses  of  growth  of  a  more  or  less  spiral 
shape  (Fig.  134).  The  liquefied  portion  gradually  tapers  off 
downwards  towards  the  needle  track.  (This  appearance  is, 
however,  in  some  varieties  not  produced  till  much  later,  especi- 
ally when  the  gelatin  is  very  stiff,  and,  in 
other  varieties  which  liquefy  very  slowly, 
may  not  be  met  with  at  all.)  At  a  later 
stage,  liquefaction  spreads  and  may  reach 
the  side  of  the  tube. 

In  gelatin  plates  the  colonies  are  some- 
what characteristic.  They  appear  as  minute 
whitish  points,  visible  in  twenty-four  to 
forty-eight  hours,  the  surface  of  which,  under 
a  low  power  of  the  miscroscope,  is  irregularly 
granular  or  furrowed  (Fig.  135,  A).  Lique- 
faction occurs,  and  the  colony  sinks  into 
the  small  cup  formed,  the  plate  then  show- 
ing small  sharply-marked  rings  around  the 
colonies.  Under  the  microscope  the  outer 
margin  of  the  cup  is  circular  and  sharply 
marked.  Within  the  cup  the  liquefied 
portion  forms  a  ring  which  has  a  more  or 
less  granular  appearance,  whilst  the  mass  of 
growth  in  the  centre  is  irregular  and  often 
broken  up  at  its  margins  (Fig.  135,  B). 

On  the  surface  of  the  agar  media  a  thin, 
almost  transparent,  layer  forms,  which  pre- 
sents no  special  characters.  On  solidified 
blood  serum  the  growth  has  at  first  the  same 
appearance,  but  afterwards  liquefaction  of 

tlle 


spirillum  in  peptone  superficial    colonies  under  a  low  power  are 

gelatin  —  six     days'  circular  discs  of  brownish-yellow  colour,  and 

growth.  Natural  size.   more  transparent  than  those  of  most  other 

organisms.       On    potato    at    the     ordinary 

temperature,  growth  does  not  take  place,  but  on  incubation  at 

a  temperature  of  from   30°  to   37°  C.,   a  moist  layer  appears, 

which  assumes  a  dirty  brown  colour,  somewhat  like  that  of  the 

glanders  bacillus  ;  the  appearance,  however,  varies  somewhat  in 

different  varieties,  and  also  on  different  sorts  of  potatoes. 

In  bouillon  with  alkaline  reaction  the  organism  grows  very 
readily,   there    occurring  in    twelve   hours  at  37°  C.  a  general 


CULTIVATION  451 

turbidity,  while  the  surface  shows  a  thin  pellicle  composed  of 
spirilla  in  a  very  actively  motile  condition.  Growth  takes  place 
under  the  same  conditions  equally  rapidly  in  peptone  solution 
(1  per  cent,  with  *5  per  cent,  sodium  chloride  added). 

In  milk  also  the  organism  grows  well,  and  produces  no 
coagulation  nor  any  change  in  its  apj>earance,  at  least  for 
several  days. 

On  all  the  media  the  growth  of  the  cholera  spirillum  is  a 
relatively  rapid  one,  and  especially  is  this  the  case  in  peptone 
solution  and  in  bouillon,  a  circumstance  of  importance  in  relation 
tn  its  separation  in  cases  of  cholera  (vide  p.  459). 

The  cholera  organism  is  one  which  grows  much  more  rapidly 
in  the  presence  of  oxygen  than  in  anaerobic  conditions ;  in  the 
complete  exclusion  of  oxygen  very  little  growth  occurs. 


A 

Kic.  !#>.—  Colonies  of  the  cholera  spirillum  in  a  gelatin  plate  — 
three  days'  growth.  A  shows  the  granular  surface,  liquefaction  just 
i  ommencing  ;  in  B  liquefaction  is  well  narked. 


I  l!"l  H'-'i-t  !<,n.  —  This  is  one  of  the  most  important  tests 
in  the  diagnosis  of  the  cholera  organism.  It  is  always  given  by 
a  true  cholera  spirillum,  and  though  the  reaction  is  not  peculiar 
to  it,  the  number  of  organisms  which  give  the  reaction  under 
the  conditions  mentioned  are  comparatively  few.  The  test  is 
made  by  adding  a  few  drops  of  pure  sulphuric  acid  to  a  culture 
in  bouillon  or  in  peptone  solution  (1  per  cent.)  which  has  been 
incubated  for  twenty-four  hours  at  37°  C.  ;  in  the  case  of  the 
cholera  spirillum  a  reddish-pink  colour  is  produced.  This  is  due 
to  the  tact  that  both  indol  and  a  nitrite  are  formed  by  the 
spirillum  in  the  medium,  and  hence  in  applying  the  test  for 
indol  tin-  addition  of  a  nitrite  is  not  necessarily  the  red  colour. 
Here,  as  in  testing  for  the  production  of  indol  by  other  bacteria. 
it  is  found  that  not  every  specimen  of  peptone  is  suitable,  and 
it  is  advisable  to  select  a  peptone  which  gives  the  characteristic 


452  CHOLERA 

reaction  with  a  known  cholera  organism,  and  to  use  it  for 
further  tests.  It  is  also  essential  that  the  sulphuric  acid  should 
be  pure,  for  if  traces  of  nitrites  are  present  the  reaction  may 
be  given  by  an  organism  which  has  not  the  power  of  forming 
nitrites. 

Htetnolytic  Test. — This  method,  introduced  by  Kraus,  is  per- 
formed by  means  of  agar  plates,  a  small  quantity  of  sterile 
defibrinated  blood  being  added  to  the  agar  and  thoroughly 
diff used ;  if  any  organism  has  haemolytic  properties,  a  clear  zone 
or  areola  forms  around  each  colony  by  the  diffusion  of  haemo- 
globin. In  no  instance  has  an  undoubted  cholera  organism 
been  found  to  produce  haemolysis,  whereas  many  species  of 
spirilla  closely  resembling  it  possess  marked  hiemolytic  action. 
This  test  may  accordingly  be  applied  along  with  the  others  in 
determining  the  identity  of  a  supposed  cholera  organism. 

Powers  of  Resistance. — In  their  resistance  against  heat, 
cholera  spirilla  correspond  with  most  spore-free  organisms,  and 
are  killed  in  an  hour  by  a  temperature  of  55°  C.,  and  much 
more  rapidly  at  higher  temperatures.  They  have  comparatively 
high  powers  of  resistance  against  great  cold,  and  have  been 
found  alive  after  being  exposed  for  several  hours  to  the  tempera- 
ture of  —  10°  G.  They  are,  however,  killed  by  being  kept  in  ice 
for  a  few  days.  Against  the  ordinary  antiseptics  they  have 
comparatively  low  powers  of  resistance,  and  Pfuhl  found  that 
the  addition  of  lime,  in  the  proportion  of  1  per  cent.,  to  water 
containing  the  cholera  organisms  was  sufficient  to  kill  them  in 
the  course  of  an  hour. 

As  regards  the  powers  of  resistance  in  ordinary  conditions, 
the  following  facts  may  be  stated  :  In  cholera  stools  kept  at  the 
ordinary  room  temperature,  the  cholera  organisms  are  rapidly 
outgrown  by  putrefactive  bacteria,  but  in  exceptional  cases  they 
have  been  found  alive  even  after  two  or  three  months.  In  most 
experiments,  however,  attempts  to  cultivate  them  even  after  a 
much  shorter  time  have  failed.  The  general  conclusion  may  be 
drawn  from  the  work  of  various  observers,  that  the  spirilla  do 
not  multiply  freely  in  ordinary  sewage  water,  although  they  may 
remain  alive  for  a  considerable  period  of  time.  On  moist  linen, 
as  Koch  showed,  they  can  nourish  very  rapidly.  Though  we 
can  state  generally  that  the  conditions  favourable  for  the  growth 
of  the  cholera  spirillum  are  a  warm  temperature,  moisture,  a 
good  supply  of  oxygen,  and  a  considerable  proportion  of  organic 
material,  we  do  not  know  the  exact  circumstances  under  which 
it  can  nourish  for  an  indefinite  period  of  time  as  a  saprophyte. 
The  fact  that  the  area  in  which  cholera  is  an  endemic  disease  is 


EXPERIMENTAL  INOCULATION  453 

so  restricted,  tends  to  show  that  the  conditions  for  a  prolonged 
UTO  \\tli  nf  tin-  spirillum  outside  the  body  are  not  usually  supplied. 
Yrt.  on  tin-  other  hand,  there  is  no  doubt  that  in  ordinary 
conditions  it  can  live  a  sufficient  time  outside  the  body  and 
multiply  t<>  a  sufficient  extent  to  explain  all  the  facts  known 
with  regard  to  the  persistence  and  spread  of  cholera  epidemics. 
During  an  epidemic  at  St.  Petersburg  the  cholera  organism 
was  cultivated  from  the  stools  of  a  considerable  number  of 
people  suffering^  f  rom  slight  intestinal  disturbance,  and  even 
(piite  healthy  individuals.  The  latter  may  be  regarded  as 
"cholera-carriers,"  but  the  organisms  were  obtained  from  them 
over  a  comparatively  short  period  of  time,  and  it  is  unknown  to 
what  extent  they  maintain  and  spread  the  infection. 

Cholera  organisms  are,  as  a  rule,  rapidly  killed  by  being 
thoroughly  dried,  and  it  is  inferred  from  this  that  they  cannot 
be  carried  in  the  living  condition  for  any  great  distance  through 
the  air,  a  conclusion  which  is  well  supported  by  observations 
on  the  spread  of  the  disease.  Cholera  is  practically  always 
transmitted  by  means  of  water  or  food  contaminated  by  the 
organism,  and  there  is  no  doubt  that  contamination  of  the 
water  supply  by  choleraic  discharges  is  the  chief  means  by  which 
areas  of  population  are  rapidly  infected.  It  has  been  shown 
that  if  Hies  are  fed  on  material  containing  cholera  organisms, 
the  organisms  may  be  found  alive  within  their  bodies  twenty- 
four  hours  afterwards.  And  further,  Haffkine  found  that 
sterilised  milk  might  become  contaminated  with  cholera 
organisms  if  kept  in  open  jars  to  which  flies  had  free  access, 
in  a  locality  infected  by  cholera.  It  is  quite  possible  that 
infection  may  be  carried  by  this  agency  in  some  cases. 

Experimental  Inoculation. — In  considering  the  effects  of 
inoculation  with  the  cholera  organism,  we  are  met  with  the 
difficulty  that  none  of  the  lower  animals,  so  far  as  is  known, 
suffer  from  the  disease  under  natural  conditions.  And,  further, 
attempts  to  produce  the  disease  by  feeding  with  cholera  dejecta, 
as  well  as  with  cultures,  have  been  unsuccessful.  As  the 
organisms  are  confined  to  the  alimentary  tract  in  the  natural 
disease,  attempts  to  induce  their  multiplication  within  the 
intestine  of  animals  by  artificially  arranging  favouring  con- 
ditions, have  occupied  a  prominent  place  in  the  experimental 
work.  \Ve  shall  give  a  short  account  of  such  experiments  : — 

Xikati  and  Rietsch  were  the  first  to  inject  the  organisms  directly  into 
tin  duodenum  of  dogs  and  rabbits,  and  they  succeeded  in  producing,  in 
a  considerable  proportion  of  the  animals,  a  choleraic  condition  of  the 
inti'.stine.  These  experiments  were  confirmed  by  other  observers,  in- 


454  CHOLERA 

eluding  Koch.  Thinking  that  probably  the  spirillum,  when  introduced 
by  the  mouth,  is  destroyed  by  the  action  of  the  hydrochloric  acid  of 
the  gastric  secretion,  Koch  first  neutralised  this  acidity  by  administering 
to  guinea-pigs  5  c.c.  of  a  5  per  cent,  solution  of  carbonate  of  soda,  and 
some  time  afterwards  introduced  a  pure  culture  into  the  stomach  by 
means  of  a  tube.  As  this  method  failed  to  give  positive  results,  he 
tried  the  effect  of  artificially  interfering  with  the  intestinal  peristalsis 
by  injecting  tincture  of  opium  into  the  peritoneum  (1  c.c.  per  200  grm. 
Aveight),  in  addition  to  neutralising  as  before  with  the  carbonate  of 
sodium  solution.  The  result  was  remarkable,  as  thirty  out  of  thirty- 
five  animals  treated  died  with  symptoms  of  general  prostration  and 
collapse.  Death  occurs  after  a  few  hours.  Post  mortem  the  small 
intestine  is  distended,  its  mucous  membrane  congested,  and  it  contains 
a  colourless  fluid  with  small  flocculi  and  the  cholera  organisms  in 
practically  pure  cultures.  Koch,  however,  found  that  when  the  spirilla 
of  Finkler  and  Prior,  of  Deneke,  and  of  Miller  (vide  infra),  were 
employed  by  the  same  method,  a  certain,  though  much  smaller, 
proportion  of  the  animals  died  from  an  intestinal  infection.  Though 
the  changes  in  these  cases  were  not  so  characteristic,  they  were  sufficient 
to  prevent  the  results  obtained  with  the  cholera  organism  from  being 
used  as  a  demonstration  of  the  specific  relation  of  the  latter  to  the 
disease. 

Within  later  years  some  additional  facts  of  high  interest  have  been 
established  with  regard  to  choleraic  infection  of  animals.  For  example, 
Sabolotny  found  that  in  the  marmot  an  intestinal  infection  readily  takes 
place  by  simple  feeding  with  the  organism,  there  resulting  the  usual 
intestinal  changes,  sometimes  with  hjemorrhagic  peritonitis  —  the 
organisms,  however,  being  present  also  in  the  blood.  And  of  special 
interest  is  the  fact,  discovered  by  Metchnikoff,  that  in  the  case  of 
young  rabbits  shortly  after  birth  a  large  proportion  die  of  choleraic 
infection  when  the  organisms  are  simply  introduced  along  with  the 
milk,  as  may  be  done  by  infecting  the  teats  of  the  mother.  Further, 
from  these  animals  thus  infected  the  disease  may  be  transmitted  to 
others  by  a  natural  mode  of  infection.  In  this  affection  of  young  rabbits 
many  of  the  symptoms  of  cholera  are  present.  Many  of  these  experi- 
ments were  performed  with  the  vibrio  of  Massowah,  which  is  now 
admitted  not  to  be  a  true  cholera  organism,  others  with  a  cholera 
vibrio  obtained  from  the  water  of  the  Seine. 

It  will  be  seen  from  the  above  account  that  the  evidence 
obtained  from  experiments  on  intestinal  infection  of  animals, 
though  by  no  means  sufficient  to  establish  the  specific  relation- 
ship of  the  cholera  organism,  is  on  the  whole  favourable  to  this 
view,  especially  when  it  is  borne  in  mind  that  animals  do  not  in 
natural  conditions  suffer  from  the  disease. 

Experiments  performed  by  direct  inoculation  also  supply 
interesting  facts.  Intraperitoneal  injection  in  guinea-pigs  is 
followed  by  general  symptoms  of  illness,  the  most  prominent 
being  distension  of  the  abdomen,  subnormal  temperature,  and, 
ultimately,  profound  collapse.  There  is  peritoneal  effusion, 
which  may  be  comparatively  clear,  or  may  be  somewhat  turbid 


EXPERIMENTS  ON  THE  HUMAN  SUBJECT       455 

and  contain  flakes  of  lymph,  according  to  the  stage  at  which 
death  takes  place.  If  the  dose  is  large,  organisms  are  found 
in  considerable  numbers  in  the  blood  and  also  in  the  small 
intestine,  but  with  smaller  doses  they  are  practically  confined  to 
the  peritoneum.  Kolle  found  that  when  the  minimum  lethal 
dose  was  used  in  guinea-pigs,  the  peritoneum  might  be  free  from 
living  organisms  at  the  time  of  death,  the  fatal  result  having 
taken  place  from  an  intoxication  (cf.  Diphtheria,  p.  405).  These 
and  other  experiments  show  that  though  the  organisms  undergo 
a  certain  amount  of  multiplication  when  introduced  by  the 
channels  mentioned,  still  the  tendency  to  invade  the  tissues  is 
not  a  marked  one.  On  the  other  hand,  the  symptoms  of  general 
intoxication  are  always  pronounced. 

Experiments  on  the  Human  Subject. — Experiments  have  also 
been  performed -in  the  case  of  the  human  subject,  both  intention- 
ally and  accidentally  In  the  course  of  Koch's  earlier  work,  one 
of  the  workers  in  his  laboratory  shortly  after  leaving  was  seized 
with  severe  choleraic  symptoms.  The  stools  were  found  to 
contain  cholera  spirilla  in  enormous  numbers.  Recovery,  how- 
ever, took  place.  In  this  case  there  was  no  other  possible 
source  of  infection  than  the  cultures  with  which  the  man  had 
been  working,  as  no  cholera  was  present  in  Germany  at  the  time. 
AVithin  recent  years  a  considerable  number  of  experiments  have 
been  performed  on  the  human  subject,  which  certainly  show  that 
in  some  cases  more  or  less  severe  choleraic  symptoms  may  follow 
ingestion  of  pure  cultures,  whilst  in  others  no  effects  'may  result. 
The  former  was  the  case,  for  example,  with  Emmerich  and 
Pettenkofer,  who  made  experiments  on  themselves,  the  former 
especially  becoming  seriously  ill.  In  the  case  of  both,  diarrhoea 
was  well  marked,  and  numerous  cholera  spirilla  were  present  in 
the  stools,  though  toxic  symptoms  were  proportionately  little 
pronounced.  Metchnikoff  also,  by  experiments  on  himself  and 
others,  obtained  results  which  convinced  him  of  the  specific 
relation  of  the  cholera  spirillum  to  the  disease.  Lastly,  we  may 
mention  the  case  of  Dr.  Orgel  in  Hamburg,  who  contracted  the 
disease  in  the  course  of  experiments  with  the  cholera  and  other 
spirilla,  and  died  in  spite  of  treatment.  It  is  believed  that  in 
sucking  up  some  peritoneal  fluid  containing  cholera  spirilla,  a 
little  entered  his  mouth  and  thus  infection  was  produced.  This 
took  place  in  September  1894,  at  a  time  when  there  was  no 
cholera  in  Germany.  On  the  other  hand,  in  many  cases  the 
experimental  ingestion  of  cholera  spirilla  by  the  human  subject 
has  given  negative  results.  Still,  as  the  result  of  observation  of 
what  takes  place  in  a  cholera  epidemic  and  of  what  has  been 


456  CHOLERA 

established  with,  regard  to  cholera  carriers,  we  may  consider 
that  only  a  certain  proportion  of  people  are  susceptible  to 
cholera,  and  the  facts  just  mentioned  are,  in  our  opinion,  of  the 
greatest  importance  in  establishing  the  relation  of  the  organism 
to  the  disease. 

Toxins. — The  general  statement  may  be  made  that  filtered 
cholera  cultures  as  a  rule  have  little  toxic  action ;  that  is,  com- 
paratively little  extracellular  toxin  is  produced  by  the  organism. 
It  was,  however,  shown  by  R.  Pfeiffer  that  the  dead  spirilla  were 
highly  toxic,  and  that,  in  fact,  they  produced,  on  injection  into 
guinea-pigs,  the  same  phenomena  as  living  cultures,  profound 
collapse  with  subnormal  temperature  being  a  prominent  feature. 
Pfeiffer  considers  that  the  toxic  substances  are  contained  in  the 
bodies  of  the  organisms — that  is,  they  are  endotoxins, — and 
that  they  are  only  set  free  by  the  disintegration  of  the 
latter.  He  showed  also  that  when  an  animal  is  inoculated 
intraperitoneally  with  the  cholera  organism,  and  then  some 
time  later  anti-cholera  serum  which  produces  bacteriolysis  is 
injected,  rapid  collapse  with  a  fatal  result  may  ensue,  apparently 
due  to  the  liberation  of  the  intracellular  toxins.  The  dead 
cultures  administered  by  the  mouth  produce  no  effect  unless 
the  intestinal  epithelium  is  injured,  in  which  case  poisoning 
may  result.  He  considers  that  the  desquamation  of  the 
epithelium  is  an  essential  factor  in  the  production  of  the 
phenomena  of  the  disease  in  the  human  subject.  Pfeiffer  found 
that  the  toxic  bodies  were  to  a  great  extent  destroyed  at  60°  C., 
but  even  after  heating  at  100°  C.  a  small  proportion  of  toxin 
remained,  which  had  the  same  physiological  action.  Later 
A.  Macfadyen  found  that  the  product  obtained  by  grinding 
up  the  spirilla  frozen  by  means  of  liquid  air  had  a  very  high 
degree  of  toxicity  when  injected  intravenously.  Like  Pfeiffer, 
he  found  that  the  "  endotoxin  "  was  in  great  part  destroyed  at 
60°  C. 

On  the  other  hand,  other  observers  (Petri,  Ransom,  Klein, 
and  others)  have  obtained  toxic  bodies  from  filtered  cultures. 
Metchnikoff,  E.  Roux,  and  Taurelli-Salimbeni  have  demon- 
strated the  formation  of  such  diffusible  toxic  bodies  in  fluid 
media.  By  means  of  cultures  placed  in  collodion  sacs  in  the 
peritoneum  of  animals,  they  found  that  the  living  organisms 
produce  toxic  bodies  which  diffuse  through  the  wall  of  the 
sac  and  cause  toxic  symptoms.  By  greatly  increasing  the 
virulence  of  the  organism,  then  growing  it  in  bouillon  and 
filtering  the  cultures  on  the  third  and  fourth  day,  they  obtained 
a  fluid  which  was  highly  toxic  to  guinea-pigs  (the  fatal  dose 


IMMUNITY  457 

usually  being  |  c.c.  per  100  gnu.  weight).  The  symptoms 
i-lust-ly  resemble  those  obtained  by  Pfeiffer.  They  found  that 
the  toxicity  of  the  filtrate  was  not  altered  by  boiling, — appar- 
ently this  toxic  substance  is  different  from  Pfeiffer's  endotoxin. 
Within  recent  times  numerous  attempts  have  been  made  to 
procure  toxic  fluids  by  disintegrating  the  cholera  spirilla,  e.g. 
by  methods  of  grinding,  by  solution  by  alkali,  by  autolysis,  etc., 
and  a  certain  measure  of  success  has  been  reached.  There  has 
also  been  much  discussion  as  to  whether  the  toxic  bodies 
obtained  are  merely  liberated  endotoxins  or  whether  they 
also  represent  true  extracellular  toxins.  The  true  relations 
of  these  bodies  has  not  yet  been  determined,  but  it  would  seem 
that  in  all  probability  the  greater  part  of  the  toxic  substance 
is  closely  bound  up  with  the  bacterial  protoplasm,  and  is  only 
set  free  on  its  disintegration. 

Immunity. — As  this  subject  is  discussed  later,  only  a  few 
facts  will  be  here  stated,  chiefly  for  the  purpose  of  making  clear 
what  follows  with  regard  to  the  means  of  distinguishing  the 
cholera  spirillum  from  other  organisms.  The  guinea-pig  or  any 
other  animal  may  be  easily  immunised  against  the  cholera 
organism  by  repeated  injections  (conveniently  made  into  the 
peritoneum)  of  non-fatal  doses  of  dead  spirilla;  later  the 
living  organisms  may  be  used.  In  this  way  a  high  degree  of 
immunity  against  the  organism  is  developed ;  and  further,  the 
blood  serum  of  an  animal  thus  immunised  (anti-cholera  serum) 
has  markedly  protective  power  when  injected,  even  in  a  small 
quantity,  into  a  guinea-pig  along  with  five  or  ten  times  the  fatal 
dose  of  the  living  organism.  Under  these  circumstances  the 
spirilla  undergo  a  granular  transformation  and,  ultimately, 
solution;  this  phenomenon  is  generally  known  as  Pfeiffer's 
reaction,  and  was  applied  by  him  to  distinguish  the  cholera 
spirillum  from  organisms  resembling  it.  The  following  are  the 
details  : — 

Pfeiffer's  Reaction.— A.  loopful  (2  mgrm.)  of  a  recent  agar  culture  of  the 
organism  to  be  tested  is  added  to  1  c.c.  of  ordinary  bouillon  containing 
'001  c.c.  of  anti-cholera  serum.  The  mixture  is  then  injected  into  the 
peritoneal  cavity  of  a  young  guinea-pig  (about  200  gnu.  in  weigbt),  and 
the  peritoneal  fluid  of  this  animal  (conveniently  obtained  by  means  of 
capillary  glass  tubes  inserted  into  the  peritoneum)  is  examined  micro- 
scopically alter  a  few  minutes.  If  the  spirilla  injected  have  been  cholera 
spirilla,  it  will  be  found  that  they  become  motionless,  swell  up  into 
globules,  and  ultimately,  break  down  and  disappear — positive  result.  If 
they  are  found  active  and  motile,  then  the  possibility  of  their  being 
true  cholera  spirilla  may  be  excluded — negative  result.  In  the  former 
case  (positive  result)  there  is,  however,  still  the  possibility  that  the 
organism  is  devoid  of  pathogenic  properties  and  has  been  destroyed  by 


458  CHOLERA 

the  normal  peritoneal  fluid.  A  control  experiment  should  accordingly 
be  made  with  '001  c.c.  of  normal  serum  in  place  of  the  anti-cholera  serum. 
If  no  alteration  of  the  organism  occurs  with  its  use,  then  the  conclu- 
sion is  that  a  true  reaction  has  been  given.  Corresponding  bacteriolytic 
effects  may  be  obtained  by  in  vitro  methods,  introduced  since  Pfeiffer's 
original  method  (p.  534). 

The  serum  of  an  animal  immunised  by  the  above  method  has 
also  marked  agglutinative  and  other  anti-bacterial  properties 
(p.  541)  against  the  cholera  spirillum,  and  these  properties  closely 
correspond  with  Pfeiffer's  reaction  as  regards  specificity.  Such 
a  serum  has,  however,  little  protective  effect  against  the  toxic 
action  of  the  dead  spirilla,  and  Pfeiffer  maintains  that  little  or 
no  antitoxin  to  the  endotoxin  can  be  produced.  On  the  other 
hand,  Macfadyen,  by  injecting  the  endotoxin  derived  from  the 
spirilla  by  grinding,  obtained  a  serum  which  had  antitoxic  as  well 
as  agglutinative  and  bacteriolytic  properties  (vide  Immunity). 
Metchnikoff  and  others  have  also  obtained  antitoxic  sera  which 
act  on  the  extracellular  toxins  obtained  by  filtration. 

The  serum  of  cholera  convalescents  has  been  found  to  possess 
protective  and  increased  bactericidal  action.  These  properties  of 
the  serum  may  be  present  eight  or  ten  days  after  the  attack  of 
the  disease,  but  are  most  marked  four  weeks  after ;  they  then 
gradually  diminish.  Specific  agglutinative  properties  have,  how- 
ever, been  detected  in  the  serum  of  cholera  patients  at  a  much 
earlier  date,  in  some  cases  even  on  the  first  day  of  the  disease, 
though  usually  a  day  or  two  later.  The  dilutions  used  have 
been  usually  1  :  15  to  1  :  120,  and  these  had  no  appreciable 
effect  on  organisms  other  than  the  cholera  spirillum  (Achard  and 
Bensande).  In  some  cases,  however,  the  agglutinative  property 
may  not  appear  after  recovery.  Variations  in  the  opsonic  index, 
analogous  to  those  in  other  diseases,  have  recently  been  observed 
in  cholera,  a  marked  fall  on  the  acute  onset  of  the  disease  being 
a  noteworthy  feature. 

Within  recent  times  there  have  been  introduced  for  therapeutic 
purposes  several  so-called  anti-sera  which  are  supposed  to  be  anti- 
toxic as  well  as  anti-bacterial,  and  of  these  the  two  most  ex- 
tensively used  are  those  of  Kraus  and  SchurupofF.  Reports 
regarding  the  effects  of  these  sera  during  the  Russian  epidemic 
are  of  somewhat  conflicting  character,  but  in  any  case  it  cannot 
be  said  that  they  have  a  markedly  beneficial  action.  They  have 
further  been  critically  examined  by  others,  who  deny  to  them  any 
marked  antitoxic  action  when  tested  experimentally.  While, 
therefore,  it  may  be  admitted  that  antitoxins  to  some  of  the 
cholera  toxins  may  be  obtained,  yet  Pfeiffer's  position,  that 


METHODS  OF  DIAGNOSIS  459 

cholera  anti-sera  have  little  effect  on  at  least  most  of  the  endo- 
toxins,  cannot  1x3  said  to  be  shaken.  It  should  be  noted,  how- 
ever, that  he  disclaims  having  made  the  general  statement,  often 
ascribed  to  him,  that  no  antitoxins  are  formed  to  endotoxins. 

Anti-Cholera  Inoculation. — Haffkine's  method  for  inoculation 
against  cholera  exemplifies  the  above  principles.  It  depends 
upon  (a)  attenuation  of  the  virus — that  is,  the  cholera  organism, 
and  (b)  exaltation  of  the  virus.  The  virulence  of  the  organism 
is  diminished  by  passing  a  current  of  sterile  air  over  the  surface 
of  the  cultures,  or  by  various  other  methods.  The  virulence  is 
exalted  by  the  method  of  passage — that  is,  by  growing  the 
organism  in  the  peritoneum  in  a  series  of  guinea-pigs.  By  the 
latter  method  the  virulence  after  a  time  is  increased  twenty-fold 
— that  is,  the  fatal  dose  has  been  reduced  to  a  twentieth  of  the 
original.  Cultures  treated  in  this  way  constitute  the  virus  exalte. 
Subcutaneous  injection  of  the  virus  exalte  produces  a  local 
necrosis,  and  may  be  followed  by  the  death  of  the  animal,  but  if 
the  animal  be  treated  first  with  the  attenuated  virus,  the  sub- 
sequent injection  of  the  virus  exalte  produces  only  a  local  oedema. 
After  inoculation  first  by  attenuated  and  afterwards  by  exalted 
virus,  the  guinea-pig  has  acquired  a  high  degree  of  immunity,  and 
Haffkine  believed  that  this  immunity  was  effective  in  the  case 
of  every  method  of  inoculation — that  is,  by  the  mouth  as  well  as 
by  injection  into  the  tissues.  After  trying  his  method  on  the 
human  subject  and  finding  it  free  from  risk,  he  extended  it 
in  practice  on  a  large  scale  in  India  in  1894.  In  the  human 
subject  two  or  sometimes  three  inoculations  were  formerly  made 
with  attenuated  virus  before  the  virus  exalte"  was  used ;  now, 
however,  a  single  injection  of  the  latter  is  usually  practised. 
The  results  of  preventive  inoculation  in  India  and  also  during 
the  recent  epidemic  in  Russia  have  been  such  as  to  establish  its 
efficiency,  both  the  case  incidence  and  the  mortality  being 
reduced. 

Methods  of  Diagnosis. — In  the  first  place,  the  stools  ought 
to  be  examinod  microscopically.  Dried  film  preparations  should 
be  made  and  stained  by  any  ordinary  stains,  though  carbol-fuchsiu 
diluted  four  times  with  water  is  specially  to  be  recommended. 
Hanging-drop  preparations,  with  or  without  the  addition  of  a 
weak  watery  solution  of  gentian-violet  or  other  stain,  should  also 
be  made,  by  which  method  the  motility  of  the  organism  can  be 
readily  seen.  By  microscopic  examination  the  presence  of  spirilla 
will  be  ascertained,  and  an  idea  as  to  their  number  obtained. 
In  some  cases  the  cholera  spirilla  are  so  numerous  in  the  stools 
that  a  picture  is  presented  which  is  obtained  in  no  other  con- 


460  CHOLERA 

dition,  and  a  microscopic  examination  may  be  sufficient  for 
practical  purposes.  According  to  Koch,  a  diagnosis  was  made 
in  50  per  cent,  of  the  cases  during  the  Hamburg  epidemic  by 
microscopic  examination  alone.  In  the  case  of  the  first  appear- 
ance of  a  cholera- like  disease,  however,  all  the  other  tests 
should  be  applied  before  a  definite  diagnosis  of  cholera  is  made. 
Dunbar  has  recently  introduced  a  method  for  rapid  diagnosis 
which  depends  on  the  properties  of  an  anti-cholera  serum.  Two 
hanging-drop  preparations  are  made,  each  consisting  of  a  small 
portion  of  mucus  from  the  suspected  stool  broken  up  in  peptone 
solution.  To  one  a  drop  of  a  50-fold  dilution  of  normal  serum 
is  added,  to  the  other  a  drop  of  a  500-fold  dilution  of  an  active 
cholera  serum.  If  the  spirilla  present  are  cholera  organisms, 
they  retain  their  motility  in  the  first  preparation,  while  they  lose 
it  and  then  become  agglutinated  in  the  second.  By  this  method 
a  diagnosis  may  sometimes  be  given  in  a  few  minutes. 

If  the  organisms  are  very  numerous,  gelatin  or  agar  plates 
may  be  made  at  once  and  pure  cultures  obtained. 

If  the  spirilla  occur  in  comparatively  small  numbers,  the  best 
method  is  to  inoculate  peptone  solution  (1  per  cent.)  and  incubate 
for  from  eight  to  twelve  hours.  At  the  end  of  that  time  the  spirilla 
will  be  found  on  microscopic  examination  in  enormous  numbers 
at  the  surface,  and  thereafter  plate  cultures  can  readily  be  made. 
If  the  spirilla  are  very  few  in  number,  or  if  a  suspected  water  is 
to  be  examined  for  cholera  organisms,  the  peptone  solution 
which  has  been  inoculated  should  be  examined  at  short  intervals 
till  the  spirilla  are  found  microscopically.  A  second  flask  of 
peptone  solution  should  then  be  inoculated,  and  possibly  again 
a  third  from  the  second,  and  then  plates  may  be  made.  In  such 
circumstances  Dieudonne's  medium  (p.  44)  has  been  found  of 
much  service. 

When  a  spirillum  has  been  obtained  in  pure  condition  by 
these  methods,  the  appearance  of  the  colonies  in  plates  should 
be  specially  noted,  the  test  for  the  cholera-red  reaction  should 
be  applied,  and  in  many  cases  it  is  advisable  to  test  the  effects 
of  intraperitoneal  injection  of  a  portion  of  a  recent  agar  culture 
in  a  guinea-pig,  the  amount  sufficient  to  cause  death  being  also 
ascertained.  The  agglutinating  or  sedimenting  properties  of  the 
serum  of  the  patient  should  be  tested  against  a  known  cholera 
organism,  and  against  the  spirillum  cultivated  from  the  case. 
The  action  of  an  anti-cholera  serum,  i.e.  the  serum  of  an  animal 
immunised  against  the  cholera  spirillum,  should  be  tested  in  a 
similar  manner. 

Up  till  recent  times  there  had  been  cultivated,  from  sources 


GENERAL  SUMMARY  461 

other  than  cholera  cases,  no  organism  which  gave  all  the  cultural 
and  biological  tests  (agglutination  and  Pfeiffer's  reaction)  of  the 
cholera  spirillum.  In  1905,  however,  Gotschlich  obtained  six 
different  strains  of  a  spirillum  which  conformed  in  all  these 
respects.  The  organisms  were  obtained  at  El  Tor  from  the 
intestines  of  pilgrims  uho  had  died  with  dysenteric  symptoms, 
and  tliciv  \\viv  no  cases  of  cholera  in  the  vicinity.  The  organisms 
in  question,  however,  differ  In  mi  the  cholera  organism  in  having 
marked  ha-niolvtic  action,  and  also  in  producing  a  rapidly  acting 
extracellular  toxin.  Kraus  and  others  have  found,  on  comparing 
anti-sera  to  the  cholera  and  El  Tor  spirilla,  that  while  the  anti- 
bacterial properties  are  similar  there  is  a  difference  in  antitoxic 
action.  The  El  Tor  antitoxin  neutralises  the  cholera  toxin,  but 
a  cholera  antitoxin  has  no  effect  on  the  El  Tor  toxin ;  the  El  Tor 
spirillum  is  thus  peculiar  as  regards  its  toxic  products.  There 
is  accordingly  difference  of  opinion  as  to  whether  these  organisms 
are  to  be  regarded  as  a  distinct  species  or  as  true  cholera  spirilla 
which  had  been  carried  by  the  patients,  though  no  symptoms 
resulted  from  their  presence.  This  instance  exemplifies  well 
the  great  difficulty  which  may  surround  the  identification  of  a 
particular  organism  obtained  from  non-cholera  cases,  and  one 
can  hardly  doubt  that  if  cholera-like  symptoms  had  been  present 
in  the  El  Tor  cases,  the  spirilla  would  have  been  accepted  as 
varieties  of  the  cholera  organism.  None  of  the  facts  ascertained, 
however,  really  affect  the  question  as  to  the  causal  relationship 
of  Koch's  spirillum  to  cholera,  although  they  indicate  the 
difficulties  which  may  attend  the  bacteriological  diagnosis  in 
isolated  cases  of  disease. 

General  Summary. — We  may  briefly  summarise  as  follows 
the  facts  in  favour  of  Koch's  spirillum  being  the  cause  of  cholera : 
First,  there  is  the  constant  presence  of  spirilla  in  true  cases  of 
cholera,  which  on  the  whole  conform  closely  with  Koch's 
description,  though  variations  undoubtedly  occur.  Moreover, 
the  facts  known  with  regard  to  their  conditions  of  growth,  etc., 
are  in  conformity  with  the  origin  and  spread  of  cholera  epidemics. 
Secondly,  the  experiments  on  animals  with  Koch's  spirillum  or 
its  toxins  give  ;is  definite  results  as  one  can  reasonably  look  for 
in  view  of  the  fact  that  animals  do  not  suffer  naturally  from  the 
disease.  Thirdly,  the  experiments  on  the  human  subject  and 
the  results  of  accidental  infection  by  means  of  pure  cultures  are 
also  strongly  in  favour  of  this  view.  Fourthly,  the  agglutinative 
and  protective  properties  of  the  serum  of  cholera  patients  and 
convalescents  constitute  another  point  in  its  favour.  Fifthly, 
bacteriological  methods,  which  proceed  on  the  assumption  that 


462  CHOLERA 

Koch's  spirillum  is  the  cause  of  the  disease,  have  been  of  the 
greatest  value  in  the  diagnosis  of  the  disease.  And  lastly,  the 
results  of  Haffkine's  method  of  preventive  inoculation  in  the 
human  subject,  which  are  on  the  whole  favourable,  also  supply 
additional  evidence.  If  all  these  facts  are  taken  together,  we 
consider  the  conclusion  must  be  arrived  at  that  the  growth  of 
Koch's  spirillum  in  the  intestine  is  the  immediate  cause  of  the 
disease.  This  does  not  exclude  the  probability  of  an  important 
part  being  played  by  conditions  of  weather  and  locality,  though 
such  are  very  imperfectly  understood.  Pettenkofer,  for  example, 
recognised  two  main  factors  in  the  causation  of  epidemics,  wrhich 
he  designated  x  and  y,  and  considered  that  these  twro  must  be 
present  together  in  order  that  cholera  may  spread.  The  or  is  the 
direct  cause  of  the  disease — an  organism  which  he  admitted 
to  be  Koch's  spirillum ;  the  y  includes  climatic  and  local  con- 
ditions, e.g.  state  of  ground-water,  etc. 

Other  Spirilla  resembling  the  Cholera  Organism. — These 
have  been  chiefly  obtained  either  from  water  contaminated  by 
sewage  or  from  the  intestinal  discharge  in  cases  with  choleraic 
symptoms.  Some  of  them  differ  so  widely  in  their  cultural  and 
other  characters  (some,  for  example,  are  phosphorescent)  that  no 
one  would  hesitate  to  classify  them  as  distinct  species.  Others, 
however,  closely  resemble  the  cholera  organism. 

The  vibrio  berolinensis,  cultivated  by  Neisser  from  Berlin  sewage 
water,  differs  from  the  cholera  organism  only  in  the  appearance  of  its 
colonies  in  gelatin  plates,  its  weak  pathogenic  action,  and  its  giving  a 
negative  result  with  Pfeiffer's  test.  It,  however,  gives  the  cholera-red 
reaction.  The  vibrio  Danubicus,  cultivated  by  Heider  from  canal  water, 
also  differs  in  the  appearance  of  its  colonies  in  plates,  and  also  reacts 
negatively  to  Pfeiffer's  test ;  in  most  respects  it  closely  resembles  the 
cholera  organism.  Another  spirillum  (v.  Ivanoff)  was  cultivated  by 
IvanofFfrom  the  stools  of  a  typhoid  patient  after  these  had  been  diluted 
with  water.  The  organism  differed  somewhat  in  the  appearance  of  its 
colonies  and  in  its  great  tendency  to  grow  out  in  the  form  of  long 
threads,  but  Pfeiffer  found  that  it  reacted  to  his  test  in  the  same  way  as 
the  cholera  organism,  and  he  considered  that  it  was  really  a  variety  of 
the  cholera  organism.  No  spirilla  could  be  found  microscopically  in  the 
stools  in  this  case,  and  Pfeiffer  is  of  the  opinion  that  the  organism 
gained  entrance  accidentally.  These  examples  will  show  how  differences 
of  opinion,  even  amongst  experts,  might  arise  as  to  whether  a  certain 
spirillum  were  really  the  cholera  organism  or  a  distinct  species  resem- 
bling it. 

A  few  examples  may  also  be  given  of  organisms  cultivated 
from  cases  in  which  cholera- like  symptoms  were  present. 

The  vibrio  of  Massowah  was  cultivated  by  Pasquale  from  a  case  during 


SPIRILLA  RESEMBLING  CHOLERA  ORGANISM     463 

a  small  epidemic  of  cholera.  The  organism  so  closely  resembles  Koch's 
spirillum  that  it  was  accepted  by  several  authorities  as  the  true  cholera 
organism,  and,  as  already  stated,  Metchnikolf  produced  by  it  cholera 
symptoms  in  the  human  subject,  and  also  the  cholera-like  disease  in 
young  rabbits.  It  possesses  four  flagella,  has  a  high  degree  of  virulence, 
producing  septicnenna  both  in  guinea-pigs  and  pigeons,  and  its  colonies 
in  plates  diller  somewhat  from  the  cholera  organism.  Moreover,  it 
reacts  negatively  to  Pfeifl'er's  test.  Another  organism,  the  v.  (Undha, 
was  cultivated  by  Pasquale  from  a  well,  and  was  at  Hrst  accepted  by 
Pfeiffer  as  the  cholera  organism,  but  afterwards  rejected,  chiefly  becaus'e 
it  failed  to  give  the  specific  immunity  reaction.  It  also  differs  somewhat 
from  the  cholera  organism  in  its  pathogenic  effects,  and  it  fails  to  give 
the  cholera-red  reaction,  or  gives  it  very  faintly. 

Pestana  and  Bettencourt  also  cultivated  a  species  of  spirillum  from  a 
number  of  cases  during  an  epidemic  in  Lisbon — an  epidemic  in  which 
there  were  symptoms  of  gastro-enteritis,  although  only  in  a  few  instances 
did  the  disease  resemble  cholera.  They  also  cultivated  the  same  organism 
from  the  drinking  water.  It  differs  from  the  cholera  organism  in  the 
appearance  of  its  colonies  and  of  puncture  cultures  in  gelatin.  It  has 
very  feeble  pathogenic  effects,  and  gives  a  very  faint,  or  no,  cholera-red 
reaction.  To  Pfeitfer's  test  it  also  reacts  negatively.  Another  spirillum 
(v.  Romanus)  was  obtained  by  Celli  and  Santori  from  twelve  out  of  forty- 
four  cases  where  there  were  the  symptoms  of  mild  cholera.  This  organism 
does  not  give  the  cholera-red  reaction,  nor  is  it  pathogenic  for  animals. 
They  look  upon  it  as  a  "transitory  variety"  of  the  cholera  organism, 
though  sufficient  evidence  for  this  view  is  not  adduced. 

We  have  mentioned  these  examples  in  order  to  show  some  of 
the  difficulties  which  exist  in  connection  with  this  subject.  It  is 
important  to  note  that,  on  the  one  hand,  spirilla  which  have 
been  judged  to  be  of  different  species  from  the  cholera  organism, 
have  been  cultivated  from  cases  in  which  cholera-like  symptoms 
were  present ;  and,  on  the  other  hand,  in  cases  of  apparently  true 
cholera  considerable  variations  in  the  characters  of  the  cholera 
organisms  have  been  found.  Such  variations  have  especially 
been  recorded  by  Colonel  Cunningham  in  India.  It  is  there- 
fore quite  an  open  question  whether  some  of  the  organisms 
in  the  former  class  may  not  be  cholera  spirilla  which  have  under- 
gone variations  as  a  result  of  the  conditions  of  their  growth. 
The  great  bulk  of  evidence,  however,  goes  to  show  that  Asiatic 
cholera  always  spreads  as  an  epidemic  from  places  in  India  where 
the  disease  is  endemic,  and  that  its  direct  cause  is  Koch's  spirillum 
with  the  characters  described  above.  It  is  sufficient  to  bear  in 
mind  that  choleraic  symptoms  may  be  produced  by  other  causes, 
and  that  in  some  of  such  cases  spirilla  which  have  a  re- 
semblance to  Koch's  orpin  ism  may  be  present  in  the  intestinal 
dis.-liarges,  though  rarely  in  large  nimiU'rs. 

A  number  of  other  spirilla  have  been  cultivated,  which  are  of 
interest  on  account  of  their  points  of  resemblance  to  the  cholera 


464  CHOLEKA 

organism,  though  probably   they  produce  no  pathological  con- 
ditions in  the  human  subject. 

Metchnikoff's  Spirillum  (vibrio  Metchnikovi).  —  This  organism  was 
obtained  by  Gamaleia  from  an  epidemic  disease  of  fowls  in  Odessa,  and 
is  of  special  interest  on  account  of  its  close  resemblance  to  the  cholera 
organism. 

Morphologically  the  organism  is  practically  identical  with  Koch's 
spirillum  (Fig.  136).  It  is  actively  motile,  and  has  the  same  staining 
reactions.  Its  growth  in  peptone-gelatin  also  closely  resembles  that  of  the 
cholera  organism,  though  it  produces  liquefaction  more  rapidly  (Fig.  137, 
A).  In  gelatin  plates  the  young  colonies  are,  however,  smoother  and  more 

circular.     After  liquefaction 

*    /~r»-      •  occurs,  some  of  the  colonies 

y  jfi  "*•£„»'  X  are  almost  identical  in 

earance  with  those 


/^'W 


ap- 

pearance with  those  of  the 
<  "  cholera  vibrio,  whilst  others 

show  more  uniformly  turbid 
contents.  In  puncture  cul- 
tures the  growth  takes  place 
more  rapidly,  but  in  appear- 
ance closely  resembles  that 
of  the  cholera  organism  a 
few  days  older.  Its  growth 
in  peptone  solution,  too,  is 
closely  similar,  and  it  also 
gives  the  cholora-red  re- 

action- 

This  organism  can,   how- 
i   (~,       **   '  ever,  be  readily  distinguished 

from   the   cholera   organism 

FIG.  136.-Metclmikoff'.s  spirillum,  both   in    ^y  the  effects  of  inoculation 

curved  and  straight  forms  ;  from  an  agar    on    animals      especially    on 

culture  of  twenty-four  hours'  growth.  pigeons     and    guinea  -  pigs. 

Stained  with  weak  carbol-fuchsin.     x  1000.      Subcutaneous  inoculation  of 

small  quantities  of  pure  cul- 
ture in  pigeons  is  followed 

by  septicaemia,  which  produces  a  fatal  result  usually  within  twenty-four 
hours.  Inoculation  with  the  same  quantity  of  cholera  culture  produces 
practically  no  result  ;  even  with  large  quantities  death  is  rarely  produced. 
The  vibrio  Metchnikovi  produces  somewhat  similar  effects  in  the  guinea- 
pig  to  those  in  the  pigeon,  subcutaneous  inoculation  being  followed  by 
extensive  hsemorrhagic  osdema  and  a  rapidly  fatal  septicaemia.  Young 
fowls  can  be  infected  by  feeding  with  virulent  cultures.  We  have 
evidence  from  the  work  of  Gamaleia  that  the  toxins  of  this  organism 
have  somewhat  the  same  action  as  those  of  the  cholera  organism. 

The  organism  is  therefore  one  which  very  closely  resembles  the  cholera 
organism,  the  results  on  inoculating  the  pigeon  offering  the  most  ready 
means  of  distinction.  It  gives  a  negative  reaction  to  Pfeiffer's  test  —  that 
is,  the  properties  of  an  anti-cholera  serum  are  not  exerted  against  it.  It 
may  also  be  mentioned  that  an  organism  which  is  apparently  the  same 
as  the  vibrio  Metchnikovi  was  cultivated  by  Pfuhl  from  water,  and  named 
v.  Nordhafen. 

Finkler  and  Prior's  Spirillum.  —  These  observers,  shortly  after  Koch's 


FIXKLER  AND  PRIOR'S  SPIRILLUM 


465 


discovery  of  the  cholera  organism,  separated  a  spirillum,  in  a  case  of 
cholera  nostras,  from  the  stools  after  they  had  been  allowed  to  decompose 
for  several  days.  There  is,  however,  no  evidence  that  the  spirillum  has 
any  causal  relationship  to  this  or  any  other  disease  in  the  human  subject. 
Morphologically  it  closely  resembles  Koch's  spirillum,  and  cannot  be 
distinguished  from  it  by  its  microscopical  characters,  although,  on  the 
whole,  it  tends  to  be  rather  thicker  in  the  centre  and  more  pointed  at  the 
ends  (Fig.  138).  In  cultures,  how- 
ever, it  presents  marked  differences. 
In  puncture  cultures  on  gelatin  it 
grows  much  more  quickly,  and  lique- 
faction is  generally  visible  within 
twenty- four  hours.  The  liquefaction 
spreads  rapidly,  and  usually  in  forty- 
eight  hours  it  has  produced  a  funnel- 
shaped  tube  with  turbid  contents, 
denser  below  (Fig.  137,  B).  In  plate 
cultures  the  growth  of  the  colonies  is 
proportionately  rapid.  Before  they 
nave  produced  liquefaction  around 
them,  they  appear,  unlike  those  of 
the  cholera  organism,  as  minute 
spheres  with  smooth  margins.  When 
liquefaction  occurs,  they  appear  MS 
little  spheres  with  turbid  contents, 
which  rapidly  increase  in  size  ;  ulti- 
mately general  liquefaction  occurs. 
On  potatoes  this  organism  grows  well 
at  the  ordinary  temperature,  and  in 
two  or  three  days  has  formed  a  slimy 
layer  of  greyish -yellow  colour,  which 
rapidly  spreads  over  the  potato.  On 
all  the  media  the  growth  has  a 
distinctly  foetid  odour.  A  growth  in 
peptone  solution  fails  to  give  the 
cholera-red  reaction  at  the  end  of 
twenty -four  hours,  though  later  a 
faint  reaction  may  appear. 

An  organism  cultivated  by  Miller 
("Miller's  Spirillum")  from  the 
cavity  of  a  decayed  tooth  in  a  human 
subject  is  almost  certainly  the  same 
organism  as  Finkler  and  Prior's 
spirillum. 

Deneke's  Spirillum— This  organ- 
ism was  obtained  from  old  cheese,  and 

is  also  known  as  the  spirillum  tyrogenum.  It  closely  resembles  Koch's 
spirillum  in  microscopic  appearances,  though  it  is  rather  thinner  and 
smaller.  Its  growth  in  gelatin  is  also  somewhat  similar,  but  liquefaction 
proceeds  more  rapidly,  and  the  bell-shaped  depression  on  the  surface  is 
larger  and  shallower,  whilst  the  growth  has  a  more  distinctly  yellowish 
tint.  The  colonies  in  plates  also  show  points  of  resemblance,  though  the 
youngest  colonies  are  rather  smoother  and  more  regular  on  the  surface, 
and  liquefaction  occurs  more  rapidly  than  in  the  case  of  the  cholera 

3° 


FIG.  137. — Puncture  cultures  in 
peptone-gelatin. 

A.  Metchnikoff's  spirillum.     Five 

days'  growth. 

B.  Finkler  and  Prior's  spirillum. 

Four  days'  growth. 
Natural  size. 


466  CHOLERA 

organism.  The  colonies  have,  on  naked-eye  examination,  a  distinctly 
yellowish  colour.  The  organism  does  not  give  the  cholera-red  reaction, 
and  on  potato  it  forms  a  thin  yellowish  layer  when  incubated  above 


x  t,    & 


!\ 


> .  V  4 


^  \' 


FIG.  138. — Fiukler  and  Prior's  spirillum  ;  from  an  agar  culture 

of  twenty-four  hours'  growth. 
Stained  with  carbol-fuchsin.     x  1000. 

30°  C.  When  tested  by  intraperitoneal  injection  and  by  other  methods, 
it  is  found  to  possess  very  feeble,  or  almost  no  pathogenic  properties. 
Deneke's  spirillum  is  usually  regarded  as  a  comparatively  harmless 
saprophyte. 


CHAPTER  XIX. 

INFLUENZA,  WHOOPING-COUGH,  PLAGUE, 
MALTA  FEVER. 


INFLUENZA. 

THE  first  accounts  of  the  organism  now  known  as  the  influenza 
bacillus  were  published  simultaneously  by  Pfeiffer,  Kitasato,  and 
Canon,  in  January  1892.  The  two  first-mentioned  observers 
found,  it  in  the  bronchial  _. 

sputum,  and  obtained  pure 


cultures,    and    Canon    ob- 
served it  in  the  blood  in  a 


i 
,' 


l 
*  .v 


• 


v 


»   T 


few  cases  of  the  disease. 
It  is,  however,  to  Pfeiffer's 
work  that  we  owe  most  of 
our  knowledge  regarding 
its  characters  and  action. 
His  results  have  been 
amply  confirmed  by  those 
of  others  in  various  epi- 
demics of  the  disease,  and 
this  organism  has  been 
generally  accepted  as  the 
cause  of  the  disease,  al- 
though absolute  proof  is 
.still  wanting. 

Microscopical  Char- 
acters.— The  influenza  bacilli  as  seen  in  the  sputum  are  very 
minute  rods  not  exceeding  1  '5  /t  in  length  and  *3  p.  in  thickness. 
They  are  straight,  with  rounded  ends,  and  sometimes  stain  more 
deeply  at  the  extremities  (Fig.  139).  The  bacilli  occur  singly 
or  form  clumps  by  their  aggregation,  but  do  not  grow  into 
chains.  They  show  no  capsule.  They  take  up  the  basic  aniline 
-tains  somewhat  feebly,  and  are  best  stained  by  a  weak  solution 

4«7 


FIG.  139. — Influenza  bacilli  from  a  culture 

on  blood  agar. 
Stained  with  carbol-t'uchsin.      x  1000. 


468  INFLUENZA 

(1  :  10)  of  carbol-fuchsin  applied  for  five  to  ten  minutes.  They 
lose  the  stain  in  Gram's  method.  They  are  non-motile,  and  do 
not  form  spores. 

In  many  cases  of  the  disease,  especially  in  the  early  stages  of 
the  more  acute,  influenza  bacilli  are  present  in  large  numbers 
and  may  be  easily  found.  On  the  other  hand,  it  is  often 
difficult  or  impossible  to  find  them,  even  when  the  symptoms 
are  severe ;  this  may  be  due  to  the  restriction  of  the  organisms 
to  some  part  not  readily  accessible,  or  it  may  be  that  they 
actually  die  out  in  great  part  while  the  effects  of  their  toxins 
persist.  It  has  also  been  observed  in  recent  epidemics,  in  which 
the  disease  has  been  less  widespread  and  on  the  whole  less 
severe,  that  the  period  during  which  the  bacilli  have  been  readily 
demonstrable  in  the  secretions  has  been  on  the  average  shorter 
than  in  the  previous  epidemics. 

Cultivation. — The  best  medium  for  the  growth  of  the 
influenza  bacillus  is  blood-smeared  agar  (see  p.  43),  which 
was  introduced  by  Pfeiffer  for  this  purpose.  He  obtained 
growths  of  the  bacilli  on  agar  which  had  been  smeared  with 
influenza  sputum,  but  he  failed  to  get  any  m6-cultures  on  the 
agar  media  or  on  serum.  The  growth  in  the  first  cultures  he 
considered  to  be  probably  due  to  the  presence  of  certain  organic 
substances  in  the  sputum,  and  accordingly  he  tried  the  expedient 
of  smearing  the  agar  with  drops  of  blood  before  making  the  in- 
oculations. In  this  way  he  completely  succeeded  in  attaining 
his  object.  The  blood  of  the  lower  animals  is  suitable,  as  well 
as  human  blood ;  and  the  favouring  influences  of  the  blood 
would  appear  to  be  due  to  the  haemoglobin,  as  a  solution  of  this 
substance  is  equally  effective.  The  colonies  of  the  influenza 
bacilli  on  blood  agar,  incubated  at  37°  C.,  appear  within  twenty- 
four  hours,  in  the  form  of  minute  circular  dots  almost  trans- 
parent, like  drops  of  dew.  When  numerous,  the  colonies  are 
scarcely  visible  to  the  naked  eye,  but  when  sparsely  arranged 
they  may  reach  the  size  of  a  small  pin's  head.  This  size  is 
generally  reached  on  the  second  day.  In  cultures  the  bacilli 
may  show  considerable  variations  in  size  and  in  shape ;  they  die 
out  somewhat  quickly,  and  in  order  to  keep  them  alive  sub- 
cultures should  be  made  every  four  or  five  days.  By  this 
method  the  cultures  may  be  maintained  for  an  indefinite  period. 
Even  in  sub-cultures  growth  on  the  ordinary  agar  media  is  slight 
and  somewhat  uncertain ;  there  is,  however,  evidence  that  growth 
is  more  marked  when  other  organisms  are  present,  that  is,  is 
favoured  by  symbiosis.  Neisser,  for  example,  was  able  to 
cultivate  the  influenza  bacillus  on  plain  agar  through  several 


DISTRIBUTION  OF  BACILLI  469 

generations  by  growing  the  xerosis  bacillus  along  with  it ;  dead 
cultures  of  the  latter  had  not  the  same  favouring  effect,  A  very 
small  amount  of  growth  takes  place  in  bouillon,  but  it  is  more 
marked  when  a  little  fresh  blood  is  added.  The  growth  forms 
a  thin  whitish  deposit  at  the  bottom  of  the  flask.  The  limits 
of  growth  are  from  25°  to  42°  C.,  the  optimum  temperature 
being  that  of  the  body.  The  influenza  bacillus  is  a  strictly 
aerobic  organism. 

The  powers  of  resistance  of  this  organism  are  of  a  low  order. 
Pfeiffer  found  that  dried  cultures  kept  at  the  ordinary  tempera- 
ture were  usually  dead  in  twenty  hours,  and  that  if  sputum 
were  kept  in  a  dry  condition  for  two  days,  all  the  influenza 
bacilli  were  dead,  or  rather,  cultures  could  be  no  longer  obtained. 
Their  duration  of  life  in  ordinary  water  is  also  short,  the  bacilli 
usually  being  dead  within  two  days.  From  these  experiments 
Pfeiffer  concludes  that  outside  the  body  in  ordinary  conditions 
they  cannot  multiply,  and  can  remain  alive  only  for  a  short 
time.  The  mode  of  infection  in  the  disease  he  accordingly 
considers  to  be  chiefly  by  means  of  fine  particles  of  disseminated 
sputum,  etc. 

Distribution  in  the  Body. — The  bacilli  are  found  chiefly  in 
the  respiratory  passages  in  influenza.  They  may  be  present  in 
large  numbers  in  the  nasal  secretion,  generally  mixed  with  a 
considerable  number  of  other  organisms,  but  it  is  in  the  small 
masses  of  greenish-yellow  sputum  from  the  bronchi  that  they 
are  present  in  largest  numbers,  in  many  cases  almost  in  a  state  of 
purity.  They  occur  in  clumps  which  may  contain  as  many  as 
100  bacilli,  and  in  the  early  stages  of  the  disease  are  chiefly 
lying  free.  As  the  disease  advances,  they  may  be  found  in 
considerable  numbers  within  the  leucocytes,  and  towards  the 
end  of  the  disease  a  large  proportion  have  this  position.  It  is 
a  matter  of  considerable  importance,  however,  that  they  may 
persist  for  weeks  after  symptoms  of  the  disease  have  disappeared, 
and  may  still  be  detected  in  the  sputum.  Especially  is  this  the 
case  when  there  is  any  chronic  pulmonary  disease.  They  also 
occur  in  large  numbers  in  the  capillary  bronchitis  and  catarrhal 
pneumonia  of  influenza,  as  Pfeiffer  showed  by  means  of  sections 
of  the  affected  parts.  In  these  sections  he  found  the  bacilli 
lying  amongst  the  leucocytes  which  filled  the  minute  bronchi, 
and  also  penetrating  between  the  epithelial  cells  and  into  the 
superficial  parts  of  the  mucous  membrane.  Other  organisms 
also,  especially  Fraenkel's  pneumococcus,  may  be  concerned  in 
the  pneumonic  conditions  following  influenza.  In  some  cases 
influenza  occurs  in  tubercular  subjects,  or  is  followed  by  tubercular 


470  INFLUENZA 

affection,  in  which  cases  both  influenza  and  tubercle  bacilli  may 
be  found  in  the  sputum.  In  such  a  condition  the  prognosis  is 
very  grave.  Regarding  the  presence  of  influenza  bacilli  in  the 
other  pulmonary  complications  following  influenza,  much  in- 
formation is  still  required.  Occasionally  in  the  foci  of  sup- 
purative  softening  in  the  lung  the  influenza  bacilli  have  been 
found  in  a  practically  pure  condition.  In  cases  of  empyema 
the  organisms  present  would  appear  to  be  chiefly  streptococci 
and  pneumococci ;  whilst  in  the  gangrenous  conditions,  which 
sometimes  occur,  a  great  variety  of  organisms  has  been  found. 

Pfeiffer's  observations  on  a  large  series  of  cases  convinced  him 
that  the  organism  was  very  rarely  present  in  the  blood — that  in 
fact  its  occurrence  there  must  be  looked  upon  as  exceptional. 
The  conclusions  of  other  observers  have,  on  the  whole,  confirmed 
this  statement,  and  it  is  probable  that 'the  chief  symptoms  in  the 
disease  are  due  to  toxins  absorbed  from  the  respiratory  tract 
(vide  infra).  In  a  recent  work,  however,  Ghedini  was  able  to 
cultivate  the  organism  from  the  blood  and  spleen  during  life  in 
over  50  per  cent,  of  the  cases  examined :  he  found  that  its 
occurrence  in  these  situations  was  specially  frequent  during 
marked  fever.  The  bacillus  may  be  present  in  some  of  the 
lesions  complicating  influenza.  Pfeiffer  found  it  in  inflammation 
of  the  middle  ear,  but  in  a  case  of  meningitis  following  influenza 
Fraenkel's  diplococcus  was  present.  In  a  few  cases  of  meningitis, 
however,  the  influenza  bacillus  has  been  found,  sometimes  alone, 
sometimes  along  with  pyogenic  cocci  (Pfulil  and  Walter,  Cornil 
and  Durante) ;  Pfulil  considers  that  in  these  the  path  of 
infection  is  usually  a  direct  one  through  the  roof  of  the  nasal 
cavity.  This  observer  also  found  post  mortem,  in  a  rapidly  fatal 
case  with  profound  general  symptoms,  influenza  bacilli  in  various 
organs,  both  within  and  outside  of  the  vessels.  In  a  few  cases 
also  the  bacilli  have  been  found  in  the  brain  and  its  membranes 
with,  little  tissue  change  in  the  parts  around. 

Extensive  observations  on  the  bacteriology  of  the  respiratory 
system  show  that  influenza-like  bacilli  may  be  present  in  a  great 
variety  of  conditions  ;  we  have,  in  fact,  once  more  to  do  with  a 
group  of  organisms  with  closely  allied  characters,  of  which 
Pfeifter's  influenza  bacillus  was  the  first  recognised  example. 
These  "  pseudo-influenza  "  bacilli  have  been  obtained  from  the 
fauces,  bronchi,  and  lungs  in  inflammatory  conditions,  and  also  in 
various  specific  fevers.  To  this  group  belongs  the  bacillus  which 
has  been  cultivated  from  cases  of  whooping-cough  by  Spengler, 
Jochmann,  Davis,  and  others,  and  which  is  present  in  consider- 
able numbers  in  a  large  proportion  of  cases  of  this  disease 


EXPERIMENTAL  INOCULATION  471 

(p.  472).  Miiller's  "trachoma  bacillus"  (p.  219)  is  a  member  of 
the  same  group.  All  these  organisms  are  very  restricted  in  their 
growth,  and  require  the  addition  of  blood  or  haemoglobin  to  the 
ordinary  culture  media ;  hence  they  are  sometimes  spoken  of  as 
hamiophilic  bacteria.  Some  of  the  examples  are  a  little  larger 
than  the  influenza  bacillus,  and  tend  to  form  short  filaments, 
but  others  are  quite  indistinguishable.  All  of  them  also  seem  to 
have  very  feeble  pathogenic  properties  towards  the  lower  animals. 
At  present  it  can  scarcely  be  claimed  as  possible  to  identify 
Pfeiffer's  bacillus  by  its  microscopic  and  cultural  characters. 

Experimental  Inoculation. --There  is  no  satisfactory  evidence 
that  any  of  the  lower  animals  suffer  from  influenza  in  natural 
conditions,  and  accordingly  we  cannot  look  for  very  definite 
results  from  experimental  inoculation.  Pfeiffer,  by  injecting 
living  cultures  of  the  organism  into  the  lungs  of  monkeys,  in 
three  cases  produced  a  condition  of  fever  of  a  remittent  type. 
There  was,  however,  little  evidence  that  the  bacilli  had  under- 
gone multiplication,  the  symptoms  being  apparently  produced 
by  their  toxins.  In  the  case  of  rabbits,  intravenous  injection  of 
living  cultures  produces  dyspnoea,  muscular  weakness,  and 
slight  rise  of  temperature,  but  the  bacilli  rapidly  disappear  in 
the  body,  and  exactly  similar  symptoms  are  produced  by 
injection  of  cultures  killed  by  the  vapour  of  chloroform. 
Pfeiffer,  therefore,  came  to  the  conclusion  that  the  influenza 
bacilli  contain  toxic  substances  which  can  produce  in  animals 
some  of  the  symptoms  of  the  disease,  but  that  animals  are  not 
liable  to  infection,  the  bacilli  not  having  power  of  multiplying 
to  any  extent  in  their  tissues. 

Cantani  succeeded  in  producing  infection  to  some  extent  in  rabbits,  by 
injecting  the  bacilli  directly  into  the  anterior  portion  of  the  brain.  In 
these  experiments  the  organisms  spread  to  the  ventricles,  and  then 
through  the  spinal  cord  by  means  of  the  central  canal,  afterwards  in- 
fecting the  substance  of  the  cord.  An  acute  encephalitis  was  thus  pro- 
duced, and  sometimes  a  purulent  condition  in  the  lateral  ventricles. 
The  bacilli  were,  however,  never  found  in  the  blood  or  in  other  organs. 
Similar  symptoms  were  also  produced  by  injection  of  dead  cultures, 
though  in  this  case  the  dose  required  to  be  five  or  six  times  larger. 
Cantani  therefore  concludes  that  the  brain  substance  is  the  most  suitable 
nidus  for  their  growth,  but  agrees  with  Pfeirl'er  in  believing  that  the 
chief  symptoms  are  produced  by  toxins  resident  in  the  bodies  ol  the  bacilli. 
He  made  control  experiments  by  injecting  other  organisms,  and  also  by 
injecting  inert  substances  into  the  cerebral  tissue. 

The  evidence,  accordingly,  that  the  influenza  bacillus  is  the 
cause  of  the  disease  rests  chiefly  on  the  well-established  fact  that 
it  is  always  present  in  the  secretions  of  the  respiratory  tract  in 


472  WHOOPING-COUGH 

true  cases  of  influenza,  and  often  in  very  large  numbers.  The 
observed  relationships  of  the  organism  to  lesions  in  the  lungs 
and  elsewhere  leave  no  room  for  doubt  that  it  is  possessed  of 
pathogenic  properties,  but  we  cannot  yet  maintain  that  its  causal 
relationship  to  epidemic  influenza  is  absolutely  established. 

Methods  of  Examination. — (a)  Microscopic. — A  portion  of  the 
greenish-yellow  purulent  material  which  often  occurs  in  little 
round  masses  in  the  sputum  should  be  selected,  and  film  prepara- 
tions should  be  made  in  the  usual  way.  Films  are  best  stained 
by  Ziehl-Neelsen  carbol-fuchsin  diluted  with  ten  parts  of  water, 
the  films  being  stained  for  ten  minutes  at  least.  In  sections  of 
the  tissues,  such  as  the  lungs,  the  bacilli  are  best  brought  out, 
according  to  Pfeiffer,  by  staining  with  the  same  solution  as  above 
for  half  an  hour.  The  sections  are  then  placed  in  alcohol 
containing  a  few  drops  of  acetic  acid,  in  which  they  are 
dehydrated  and  slightly  decolorised  at  the  same  time.  They 
should  be  allowed  to  remain  till  they  have  a  moderately  light 
colour,  the  time  varying  according  to  their  appearance.  They 
are  then  washed  in  pure  alcohol,  cleared  in  xylol,  and  afterwards 
mounted  in  balsam. 

(b)  Cultures. — A  suitable  portion  of  the  greenish-yellow 
material  having  been  selected  from  the  sputum,  it  should  be 
washed  well  in  several  changes  of  sterilised  water.  A  portion 
should  then  be  taken  on  a  platinum  needle,  and  successive 
strokes  made  on  the  surface  of  blood-agar  tubes.  The  tubes 
should  then  be  incubated  at  37°  C.,  when  the  transparent 
colonies  of  the  influenza  bacillus  will  appear,  usually  within 
twenty -four  hours.  These  should  give  a  negative  result  on 
inoculation  on  ordinary  agar  media. 

WHOOPING-COUGH. 

Up  to  the  year  1906,  the  chief  result  of  bacteriological 
observations,  of  which  those  of  Spengler,  Krause  and  Jochmann, 
and  Davis  may  specially  be  mentioned,  had  been  to  demonstrate 
the  very  frequent  presence  of  minute  influenza-like  and  haemo- 
philic  bacilli  in  the  sputum  and  also  in  the  lesions  in  this  disease. 
In  the  year  mentioned,  however,  Bordet  and  Gengou  published 
an  account  of  another  minute  organism,  and  brought  forward 
certain  facts  which  gave  strong  support  to  its  etiological 
relationship.  A  short  ^description  of  this  bacillus  may 
accordingly  be  given. 

Characters  of  the  Bacillus  (Bordet-Gengou). — The  organism, 
as  seen,  for  example,  in  the  sputum,  occurs  in  the  form  of 


CHARACTERS  OF  THE  BACILLUS 


473 


minute  oval  rods  scarcely  larger  than  the  influenza  bacillus. 
They  stain  rather  faintly  with  ordinary  stains,  and  their  margin 
and  extremities  are  often  more  deeply  coloured  than  the  centre, 
which  may  appear  as  an  uncoloured  spot;  they  are  Gram- 
negative  and  do  not  form  spores.  In  cultures  they  present  the 
same  characters  and  are  less  pleomorphous  than  the  influenza 
bacillus  (Fig.  140).  They  are  specially  numerous  at  the  beginning 
of  the  disease,  and  they  may  be  found  in  large  numbers  in  almost 
pure  culture  in  the  opaque  whitish  sputum  expectorated  from  the 
bronchi ;  as  the  disease  advances  they  become  scanty,  and  may 
disappear  when  the 
symptoms  of  the  disease 
are  still  prominent. 
Bordet  and  Gengou  suc- 
ceeded in  obtaining  pure 
cultures  on  the  blood- 
agar  medium  described 
on  p.  44,  and  this  was 
found  to  be  the  most 
suitable  of  all  the  media 
tried.  In  the  first  cul- 
tures growth  is  very 
scanty  and  may  be  in- 
visible, but  later  it 
becomes  much  more 
abundant,  and  sub-cul- 
tures may  also  be 

,.,  ,.         FIG.  140.1— Film  preparation  from  a  twenty- 

readily  made  on  ordm-       four  hours>  cuiture  of  the  bacillus  of  whoop- 
ary   serum-agar    media.        ing-cough.    (Bordet-Gengou). 
As  compared  with  that  Stained  with  carbol-fuchsin.     xlOOO. 

of  the  influenza  bacillus, 

growth  is  thicker  and  less  transparent  and  the  margins  are 
more  sharply  marked  off;  the  presence  of  haemoglobin,  though 
favouring  the  growth,  is  not  so  essential  as  in  the  case  of 
the  latter  organism.  The  organism  is  a  strict  aerobe,  and  in 
the  case  of  cultures  in  fluid  media,  e.y.  serum  bouillon,  the 
tubes  ought  to  be  placed  in  a  sloped  position,  in  order  to 
expose  a  greater  surface  to  the  air.  Bordet  and  Gengou 
completely  confirmed  the  observations  mentioned  above  as  to  the 
very  frequent,  almost  constant,  presence  of  influenza-like  bacilli. 
They  obtained  growths  of  these  organisms,  and  on  comparing 
them  with  their  own  bacillus  found  that  distinct  cultural 

1  We  are  indebted  to  Dr.  Bordet  for  the  culture  from  which  this  preparation 

was  made. 


'.  »*•  »«*  *~ 
•*»*£.  -,;,.:- 


474  WHOOPING-COUGH 

differences  could  be  made  out.  The  most  important  distinctions 
were,  however,  obtained  on  studying  the  serum  reactions  of 
convalescents  from  the  disease.  They  found  that  in  many  cases, 
though  not  invariably,  such  sera  agglutinated  their  bacillus,  but 
none  of  the  influenza-like  organisms.  The  most  important 
result,  however,  was  that  in  every  case  examined  the  serum  of 
convalescents  gave  the  deviation  of  complement  reaction  very 
markedly  with  the  whooping-cough  bacillus,  but  with  none  of  the 
others.  This  means,  of  course,  that  a  true  anti-substance  to  the 
bacillus  (immune-body  or  substance  sensibilisatrice)  was  present 
in  the  serum,  and  points  to  a  true  infection  with  the  organism 
(p.131). 

Pathogenic  Effects. — The  general  results  obtained  by 
Bordet  and  Gengou  were  that  the  ordinarily  used  animals  were 
not  susceptible  to  true  infection  with  the  bacillus,  but  that  it 
contained  a  powerfully  acting  endotoxin,  which  produced  both 
local  and  general  effects.  The  injection  of  a  small  quantity  of 
the  bacillus  into  the  eye  of  a  rabbit  produced  a  local  necrosis, 
with  little  inflammatory  change,  and  the  introduction  of  dead, 
as  well  as  living,  cultures  into  the  peritoneal  cavity  of  a  guinea- 
pig  caused  death  from  toxic  action,  there  being  great  effusion 
into  the  cavity  and  numerous  haemorrhages  in  its  lining. 

They  advanced  the  view  that  the  bacillus  is  present  in  large 
numbers  at  the  beginning  of  the  disease,  and  inflicts  some  local 
damage  on  the  bronchial  tubes  which  may  persist  after  the  dis- 
appearance of  the  bacillus  and  keep  up  the  irritation. 

Similar  results  were  obtained  with  an  endotoxin  prepared  accord- 
ing to  Besredka's  method.  It  was  not  found  possible  to  obtain  an 
anti-toxin  to  this  toxin.  Very  important  results  have,  however, 
been  since  obtained  by  Klimenko,  who  succeeded  in  infecting 
monkeys  and  young  dogs  by  intratracheal  injection  of  pure 
cultures  of  the  bacillus.  After  a  period  of  incubation,  there 
occurred  an  illness  in  which  symptoms  of  pulmonary  irritation 
and  irregular  pyrexia  were  outstanding  features.  Usually,  in 
the  case  of  the  dogs,  a  fatal  result  followed  after  two  or  three 
weeks,  and  post  mortem  there  were  found  symptoms  of  catarrh 
of  the  respiratory  tract  and  sometimes  patches  of  broncho- 
pneumonia,  from  which  the  bacillus  could  be  recovered  in  pure 
culture.  The  serum  of  the  infected  animals  gave  the  deviation 
of  complement  reaction.  A  specially  interesting  fact  is  that  a 
number  of  healthy  young  dogs  contracted  the  disease  by  contact 
with  the  inoculated.  Fraenkel  also  obtained  positive  results, 
closely  similar  to  those  of  Kliinenko,  on  inoculation  with  pure 
cultures  of  the  bacillus.  • 


METHODS  OF  EXAMINATION  475 

The  results  of  Bordet  and  Gengou  have  received  general  con- 
firmation, although  it  is  to  be  noted  that  Fraenkel  and  also 
Wollstein  failed  to  obtain  the  deviation  of  complement  reaction 
with  the  serum  of  convalescents.  Bordet  and  Gengou  have 
inquired  into  this  discrepancy  in  the  case  of  the  former,  and  find 
that  it  depends  on  the  nature  of  the  culture  medium  used.  At 
present  it  is  not  justifiable  to  make  a  definite  pronouncement  on 
the  subject.  We  can  only  say  that  Bordet  and  Gengou  have 
made  out  a  strong  case  for  the  etiological  relationship  .of  their 
bacillus,  and  that  their  observations  have  been  confirmed  by 
those  of  others. 

Methods  of  Examination. — A  portion  of  sputum  expectorated 
during  a  paroxysm  of  coughing  should  be  obtained  at  as  early  as 
possible  a  stage  of  the  disease  ;  film  preparations  should  be  made 
in  the  usual  way  and  stained  by  carbol-thionin  or  carbol-methylene 
blue.  If  the  characteristic  bacilli  largely  preponderate,  tubes  of 
the  Bordet-Gengou  medium  may  then  be  inoculated  and  in- 
cubated. If  there  are  numerous  colonies  of  other  organisms  in 
the  tubes,  a  portion  of  the  intervening  agar  should  be  scraped 
with  a  needle  and  fresh  tubes  inoculated.  As  already  said, 
growth  is  at  first  very  scanty  but  becomes  more  luxuriant  in 
sub-cultures.  On  pure  cultures  being  obtained,  the  deviation  of 
complement  test  is  to  be  applied  by  the  method  described  (p.  1 30). 

PLAGUE. 

The  bacillus  of  Oriental  plague  or  bubonic  pest  was  discovered 
independently  by  Kitasato  and  by  Yersin  during  the  epidemic 
at  Hong  Kong  in  1894.  They  cultivated  the  organism  from  a 
large  number  of  cases  of  plague,  and  reproduced  the  disease  in 
susceptible  animals  by  inoculation  of  pure  cultures.  It  is  to 
be  noted  that  during  an  epidemic  of  plague,  sometimes  even 
preceding  it,  a  high  mortality  has  been  observed  amongst  certain 
animals,  especially  rats  and  mice,  and  that  from  the  bodies  of 
these  animals  found  dead  in  the  plague-stricken  district,  the  same 
bacillus  was  obtained  by  Kitasato  and  also  by  Yersin. 

Bacillus  of  Plague — Microscopical  Characters. — As  seen  in 
the  affected  glands  or  buboes  in  this  disease,  the  bacilli  are 
small  oval  rods,  somewhat  shorter  than  the  typhoid  bacillus, 
and  about  the  same  tliirkiu-ss  (Fi«j.  Ill),  though  considerable 
variations  in  size  occur.  They  have  rounded  ends,  and  in 
stained  preparations  a  portion  in  the  middle  of  the  bacillus  is 
often  left  uncoloured,  giving  the  so-called  "  i>olar  staining."  In 
films  from"  the  tissues  they  are  found  scattered  amongst  the  cells, 


476 


PLAGUE 


for  the  most  part  lying  singly,  though  pairs  are  also  seen.  On 
the  other  hand,  in  cultures  in  fluids,  e.g.  bouillon,  they  grow 
chiefly  in  chains,  sometimes  of  considerable  length,  the  form 
known  as  a  streptobacillus  resulting  (Fig.  143).  In  young  agar 
cultures  the  bacilli  show  greater  variation  in  size,  and  polar 
staining  is  less  marked  than  in  the  tissues :  sometimes  forms 
of  considerable  length  are  present.  After  a  time  involution 


^ 


FIG.  141.  —Film  preparation  from  a  plague  bubo  showing  enormous 
numbers  of  bacilli,  most  of  which  show  well-marked  bipolar  staining. 
Stained  with  weak  gentian-violet,      x  1000. 


forms  appear,  especially  when  the  surface  of  the  agar  is  dry; 
but  the  formation  of  these  is  much  more  rapid  and  more  marked 
when  2  to  5  per  cent,  of  sodium  chloride  is  added  to  the  medium, 
constituting  the  so-called  "  salt  agar  "  (Hankin  and  Leumann). 
On  this  medium,  especially  with  the  higher  percentage,  the  in- 
volution forms  assume  a  great  size  and  a  striking  variety  of 
shapes,  large  globular,  oval,  or  pyriform  bodies  resulting  (Fig. 
144)  ;  with  about  2  per  cent,  sodium  chloride,  after  twenty- 
four  hours'  incubation,  the  most  striking  feature  is  -a  general 


CULTIVATION  OF  BACILLUS 


477 


/  ^ 

uv 


V>*^^i        J\  »  S     v     /        -*r>     t- 

fouml  w\y!>?  ',  V-~*  v'-«  x       '»§&     ' 

flagelk  .>    KVy^V'VVji    ', 
ith  ''^c*^   ,'   ..  « 

E  <#'%>W^ 

"':  vVCs^jy 

of  ^   ,  -     ^   -_    v.   .rx  ' 


enlargement  of  all  the  bacilli.     Sometimes  in  the  tissues  they 
are  seen  to  be  surrounded  _____ 

by  an  unstained   capsule, 
though  this  appearance  is 
by    no     means    common. 
They  do  not  form  spores.        ^T, 
Gordon,    who    has    found      >\^ 
that  they  possess 
which,  however,  stain  with 
difficulty,  states  that  they 
are     motile.       Most    ob- 
servers, however,  and  with 
these      we     agree,      have 
failed  to  find  evidence  ui  r  -    t  . 

true  inotility.     They  stain  *         '  **       •        H^' 

readily     with     the     basic  *  J^s~~       ' 

aniline     stains,     but     are 

,       FIG.  142.-  Bacillus  of  plague  from  a  young 

decolorised      by      Grams  culture  on  agar. 

i  lift  hod.  Stained  with  weak  carbol-fuchsiii.     x  1000. 

Cultivation.  — From  the 

atl'rcted  glands,  etc.,  the  bacillus  can  readily  be  cultivated  on 

the  ordinary  media.     It  grows  best  at  the  temperature  of  the 

body,  though  growth 
occurs  as  low  as  18°  C. 
On  agar  and  on  blood 
serum  the  colonies  are 
whitish  circular  discs  of 
somewhat  transparent 
appearance  and  smooth, 
shining  surface.  When 
examined  with  a  lens, 
their  borders  appear 
slightly  wavy.  In  stroke 
cultures  on  agar  there 
forms  a  continuous  line 
of  growth  with  the 
same  appearance,  show- 
ing partly  separated 

FIG.  143 -Bacillus  of  plague  in  chains  show-     colonies    at   its    margins, 
ing  polar  staining.     From  a  young  culture      ,I7, 

in  bouillon.  When  agar  cultures  are 

Stained  with  thionin-blue.     x  1000.          kept  at  the  room  tempera- 
ture, some  of  the  colonies 

may  show  a  more  luxuriant  growth  with  more  opaque  appearance 

than  the  rest  of  the  growth,  the  appearance  in  fact  being  often 


478  PLAGUE 

such  as  to  suggest  the  presence  of  impurities  in  the  cultures.  In 
stab  cultures  in  peptone  gelatin,  growth  takes  place  along  the 
needle  track  as  a  white  line,  composed  of  small  spherical  colonies. 
On  the  surface  of  the  gelatin  a  thin,  semi-transparent  layer  may 
be  formed,  which  is  usually  restricted  to  the  region  of  puncture, 
though  sometimes  it  may  spread  to  the  wall  of  the  tube ;  some- 
times, however,  there  is  practically  no  surface  growth.  There  is 
no  liquefaction  of  the  medium.  In  gelatin  plates  the  superficial 
colonies  develop  first  and  form  slightly  raised  semi-transparent 
discs  with  somewhat  crenated  margins ;  the  deeper  colonies  are 

smaller  and  of  spherical 
shape,  with  smooth  out- 
line. In  bouillon  the 

^  'v  growth   usually   forms    a 

%••""          slightly       granular        or 
•  v       powdery    deposit    at    the 

*  ^°°*    anc^    sides    of     the 

•  flask,     somewhat     resem- 
bling   that  of  a    strepto- 
coccus.     If  oil  or  melted 

-i, *m+  butter   is   added    to    the 

**t    %  ^"    «         V-*V"4«?I          '  \S       bouillon    so    that     drops 
-*    ***«•*          ^?£**:V*'         float  on  the  surface,  then 

a  striking  mode  of  growth 
may  result,  to  which  the 
term  "stalactite"  has  been 

FIG.  144.— Culture  of  the  bacillus  of  plague  applied.  This  consists  in 
on  4  per  cent,  salt  agar,  showing  involution  the  growth  starting  from 
forms  of  great  variety  of  size  and  shape.  ,,  11TU3PP  allrfoPP  Of  flip 
See  also  Plate  IV.,  Fig.  17.  tne  Uinclern  &™ce  01 

Stained  with  carbol-thionin-blue.      xlOOO.      fat    globules   and   extend- 
ing    downwards     in    the 

form  of  pendulous,  string-like  masses.  These  masses  are 
exceedingly  delicate,  and  readily  break  off  on  the  slightest 
shaking  of  the  flask;  accordingly  during  their  formation  the 
culture  must  be  kept  absolutely  at  rest.  This  manner  of 
growth  constitutes  an  important  but  not  absolutely  specific 
character  of  the  organism ;  unfortunately  it  is  not  supplied  by 
all  races  of  the  organism,  and  varies  from  time  to  time  with 
the  same  race.  The  organism  flourishes  best  in  an  abundant 
supply  of  oxygen ;  in  strictly  anaerobic  conditions  almost  no 
growth  takes  place. 

The  organism  in  its  powers  of  resistance  corresponds  with 
other  spore-free  bacilli,  and  is  readily  killed  by  heat,  an  exposure 
for  an  hour  at  58°  C.  being  fatal.  On  the  other  hand,  it  has 


ANATOMICAL  CHANGES  479 

remarkable  powers  of  resistance  against  cold ;  it  has  been  exposed 
to  a  temperature  several  degrees  below  freezing-point  without 
I  iri ng  killed.  Experiments  on  the  effects  of  drying  have  given 
somewhat  diverse  results,  but  as  a  rule  the  organism  has  been 
found  to  be  dead  after  being  dried  for  from  six  to  eight  days, 
though  sometimes  it  has  survived  the  process  for  a  longer  period ; 
exposure  to  direct  sunlight  for  three  or  four  hours  kills  it.  When 
cultivated  outside  the  body  the  organism  often  loses  its  virulence, 
but  some  races  remain  virulent  in  cultures  for  a  long  period  of 
time. 

Anatomical  Changes  and  Distribution  of  Bacilli. — The 
disease  occurs  in  several  forms,  the  bubonic  and  the  pulmonary 
being  the  best  recognised ;  to  these  may  be  added  the  septiccemic. 
The  most  striking  feature  in  the  bubonic  form  is  the  affection 
of  the  lymphatic  glands,  which  undergo  intense  inflammatory 
swelling,  attended  with  haemorrhage,  and  generally  ending  in 
a  greater  or  less  degree  of  necrotic  softening  if  the  patient  lives 
long  enough.  The  connective  tissue  around  the  glands  is 
similarly  affected.  The  bubo  is  thus  usually  formed  by  a 
collection  of  enlarged  glands  fused  by  the  inflammatory  swelling. 
True  suppuration  is  rare.  Usually  one  group  of  glands  is 
affected  first,  constituting  the  primary  bubo — in  the  great 
majority  the  inguinal  or  the  axillary  glands — and  afterwards 
other  groups  may  become  involved,  though  to  a  much  less 
extent.  Along  with  these  changes  there  is  great  swelling  of 
the  spleen,  and  often  intense  cloudy  swelling  of  the  cells  of  the 
kidneys,  liver,  and  other  organs.  There  may  also  occur  secondary 
areas  of  haemorrhage  and  necrosis,  chiefly  in  the  lungs,  liver, 
and  spleen.  The  bacilli  occur  in  enormous  numbers  in  the 
swollen  glands,  being  often  so  numerous  that  a  film  preparation 
made  from  a  scraping  almost  resembles  a  pure  culture  (Fig. 
141).  In  sections  of  the  glands  in  the  earlier  stages  the  bacilli 
are  found  to  form  dense  masses  in  the  lymph  paths  and  sinuses 
(Fig.  145),  often  forming  an  injection  of  them;  they  may  also 
be  seen  growing  as  a  fine  reticulum  between  the  cells  of  the 
lymphoid  tissue.  At  a  later  period,  when  disorganisation  of 
the  gland  has  occurred,  they  become  irregularly  mixed  with  the 
cellular  elements.  Later  still  they  gradually  disappear,  and 
wlp.-ii  necrosis  is  well  advanced  it  may  be  impossible  to  find  any 
— a  point  of  importance  in  connection  with  diagnosis.  In  the 
spleen  they  may  be  very  numerous  or  they  may  be  scanty, 
according  to  the  amount  of  blood  infection  which  has  occurred ; 
in  the  secondary  lesions  mentioned  they  are  often  abundant, 
lu  the  pulmonary  form  the  lesion  is  the  well-recognised  "plague 


480 


PLAGUE 


pneumonia."  This  is  of  broncho-pneumonic  type,  though  large 
areas  may  be  formed  by  confluence  of  the  consolidated  patches, 
and  the  inflammatory  process  is  attended  usually  by  much 
hemorrhage ;  the  bronchial  glands  show  inflammatory  swelling. 
Clinically  there  is  usually  a  fairly  abundant  frothy  sputum  often 
tinted  with  blood,  and  in  it  the  bacilli  may  be  found  in  large 
numbers.  Sometimes,  however,  cough  and  expectoration  may 


FIG.  145. — Section  of  a  human  lymphatic  gland  in  plague,  showing 
the  injection  of  the  lymph  paths  and  sinuses  with  masses  of  plague 
bacilli — seen  as  black  areas. 

Stained  with  carbol-thionin-blue.      x  50. 


be  absent.  The  disease  in  this  form  is  said  to  be  invariably 
fatal.  In  the  septiccemic  form  proper  there  is  no  primary  bubo 
discoverable,  though  there  is  almost  always  slight  general  en- 
largement of  lymphatic  glands;  here  also  the  disease  is  of 
specially  grave  character.  A  bubonic  case  may,  however, 
terminate  with  septicaemia ;  in  fact  all  intermediate  forms  occur. 
An  intestinal  form  with  extensive  affection  of  the  mesenteric 
glands  has  been  described,  but  it  is  exceedingly  rare — so  much 


EXPERIMENTAL  INOCULATION  481 

so  that  many  observers  with  extensive  experience  have  doubted 
its  occurrence.  In  the  various  forms  of  the  disease  the  bacilli 
occur  also  in  the  blood,  in  which  they  may  be  found  during  life 
by  microscopic  examination,  chiefly,  however,  just  before  death 
in  very  severe  and  rapidly  fatal  cases.  The  examination  of  the 
blood  by  means  of  cultivation  experiments  is,  however,  a  much 
more  reliable  procedure.  For  this  purpose  about  1  c.c.  of  blood 
may  be  withdrawn  from  a  vein  and  distributed  in  flasks  of 
bouillon  (p.  72).  It  may  be  said  from  the  results  of  different 
investigators  that  the  bacillus  may  be  obtained  by  culture  in 
fully  50  per  cent,  of  the  cases,  though  the  number  will  necessarily 
vary  in  different  epidemics.  The  Advisory  Committee,  ap- 
pointed by  the  Secretary  of  State  for  India  in  1905,  found  that 
in  some  septicuemic  cases  the  bacilli  may  be  present  in  the  blood 
in  large  numbers,  two,  or  even  three,  days  before  death,  though 
this  is  exceptional. 

The  above  types  of  the  disease  are  usually  classified  together 
under  the  heading  pestis  major,  but  there  also  occur  mild  forms 
to  which  the  term  pestis  minor  is  applied.  In  these  latter  there 
may  be  a  moderate  degree  of  swelling  of  a  group  of  glands, 
attended  with  some  pyrexia  and  general  malaise,  or  there  may 
!><•  little  more  than  slight  discomfort.  Between  such  and  the 
graver  types,  cases  of  all  degrees  of  severity  are  met  with. 

Experimental  Inoculation. — Mice,  guinea-pigs,  rats,  and 
rabbits  are  susceptible  to  inoculation,  the  two  former  being  on 
the  whole  most  suitable  for  experimental  purposes.  After  sub- 
cutaneous injection  there  occurs  a  local  inflammatory  oedema, 
which  is  followed  by  inflammatory  swelling  of  the  corresponding 
lymphatic  glands,  and  thereafter  by  a  general  infection.  The 
lesions  in  the  lymphatic  glands  correspond  in  their  main 
characters  with  those  in  the  human  subject,  although  usually 
at  the  time  of  death  they  have  not  reached  a  stage  so  advanced. 
By  this  method  of  inoculation  mice  usually  die  in  1  to  3  days, 
guinea-pigs  and  rats  in  2  to  5  days,  and  rabbits  in  4  to  7  days. 
IVst  mortem  the  chief  changes,  in  addition  to  the  glandular 
enlargement,  are  congestion  of  internal  organs,  sometimes  with 
hemorrhages,  and  enlargement  of  the  spleen ;  the  bacilli  are 
numerous  in  the  lymphatic  glands  and  usually  in  the  spleen 
(Fig.  146),  and  also,  though  in  somewhat  less  degree,  throughout 
the  blood.  Infection  can  also  be  produced  by  smearing  the 
material  on'  the  conjunctiva  or  mucous  membrane  of  the  nose, 
and  this  method  of  inoculation  has  been  successfully  applied  in 
cases  where  the  plague  bacilli  are  present  along  with  other 
virulent  organisms,  e.g.  in  sputum  along  with  pneumococci. 


482  PLAGUE 

Rats  and  mice  can  also  be  infected  by  feeding  either  with  pure 
cultures  or  with  pieces  of  organs  from  cases  of  the  disease, 
though  in  this  case  infection  probably  takes  place  through  the 
mucous  membrane  of  the  mouth  and  adjacent  parts,  and  only  to 
a  limited  extent,  if  at  all,  by  the  alimentary  canal.  Monkeys 
also  are  highly  susceptible  to  infection,  and  it  has  been  showrn 
in  the  case  of  these  animals  that  when  inoculation  is  made  on 
the  skin  surface,  for  example,  by  means  of  a  spine  charged  with 
the  bacillus,  the  glands  in  relation  to  the  part  may  show  the 
characteristic  lesion  and  a  fatal  result  may  follow  without  there 

being      any     noticeable 

-  -  «,f~*>f  jf*»    ..  lesion    at    the     primary 

*!  r*  V  **     ****  sea*-      This    fact    throws 

important  light  on  in- 
fection by  the  skin  in 
the  human  subject.  The 
disease  may  also  extens- 
ively affect  monkeys  by 
natural  means  during  an 
epidemic. 

Paths  and  Mode  of 
Infection. — Plague 
bacilli  may  enter  the  sys- 


through    small    wounds, 
cracks,     abrasions,     etc., 

FIG.  146.-Film  preparation  of  spleen  of  rat  and  in  snch  cases  there 
after  inoculation  with  the  bacillus  of  plague,  IS  usually  no  reaction 
showing  numerous  bacilli,  most  of  which  are  at  the  site  of  entrance, 
somewhat  plump.  m-,  .  ,  ,.  . 

Stained  with  carbol-thionin-blue.     x  1000.       ims     last      tact      1S      m 

accordance     with     what 

has  been  stated  above  with  regard  to  experiments  on 
monkeys.  The  path  of  infection  is  shown  by  the  primary 
buboes,  which  are  usually  in  .the  glands  through  which  the 
skin  is  drained,  those  in  the  groin  being  the  commonest  site. 
Absolute  proof  of  the  possibility  of  infection  by  the  skin  is 
supplied  by  several  cases  in  which  the  disease  has  been  acquired 
at  post  mortem  examinations,  the  lesions  of  the  skin  surface 
being  in  the  majority  of  these  of  trifling  nature ;  in  only  two 
was  there  local  reaction  at  the  site  of  inoculation.  In  most  of 
these  cases  the  period  of  incubation  has  been  from  two  to  three 
days ;  under  natural  conditions  of  infection  the  average  period 
is  within  five  days.  While  infection  may  occur  by  accidental 
inoculation  through  small  wounds  of  the  skin  surface,  it  appears 


PATHS  AND  MODE  OF  INFECTION  483 

in  the  majority  of  cases  to  take  place  by  means  of  the  bites 
of  fleas.  For  some  time  it  had  been  known  that  plague  bacilli 
might  be  found  for  some  time  afterwards  in  the  stomach  of  fleas 
allowed  to  feed  on  animals  suffering  from  plague,  and  some 
observers,  for  example  Simond,  had  succeeded  in  transmitting 
the  disease  to  other  animals  by  means  of  the  infected  insects. 
Most  observers,  however,  had  obtained  negative  results,  and  it 
was  only  by  the  work  of  the  Advisory  Committee  referred  to 
above,1  that  the  importance  of  this  means  of  infection  was  estab- 
lished. By  carefully  planned  experiments,  the  Committee  showed 
that  the  disease  could  be  transmitted  from  a  plague  rat  to  a 
healthy  rat  kept  in  adjacent  cages  when  fleas  were  present ; 
whereas  this  did  not  occur  when  means  were  taken  to  prevent 
the  access  of  fleas,  though  the  facilities  for  aerial  infection 
were  the  same.  The  disease  can  also  be  produced  by  fleas 
removed  from  plague  rats  and  transferred  directly  to  healthy 
animals,  success  having  been  obtained  in  fully  50  per  cent,  of 
experiments  of  this  kind.  When  plague-infected  guinea-pigs 
are  placed  amongst  healthy  guinea-pigs,  comparatively  few  of 
the  latter  acquire  the  disease  when  fleas  are  absent  or  scanty ; 
whereas  all  of  them  may  die  of  plague  when  fleas  are  numerous. 
This  result  demonstrates  the  comparatively  small  part  played 
by  direct  contact,  even  when  of  a  close  character.  Important 
results  were  also  obtained  with  regard  to  the  mode  of  infection 
in  houses  where  there  had  been  cases  of  plague.  It  was  found 
possible  to  produce  the  disease  in  susceptible  animals  by  means 
of  fleas  taken  from  rats  in  plague  houses.  When  animals  were 
placed  in  plague  houses  and  efficiently  protected  from  fleas  they 
remained  healthy ;  whereas  they  acquired  the  disease  when  the 
cages  were  free  to  the  access  of  fleas  in  the  neighbourhood. 

The  following  are  some  of  the  experiments  which  were  conducted  : — A 
series  of  six  huts  were  built  which  only  differed  in  the  structure  of  their 
roofs.  In  two  the  roofs  were  made  of  ordinary  native  tiles  in  which  rats 
freely  lodge  ;  in  two  others,  flat  tiles  were  used  in  which  rats  live,  but  in 
which  they  have  not  such  facilities  for  movement  as  in  the  first  set,  and 
in  the  third  pair  the  roof  was  formed  of  corrugated  iron.  Under  the 
roof  in  each  case  was  placed  a  wire  diaphragm  which  prevented  rats  or 
their  droppings  having  access  to  the  hut,  but  which  would  not  prevent 
fleas  falling  down  on  to  the  floor  of  the  hut.  The  huts  were  left  a 
sufficient  time  to  become  infected  with  rats,  and  then  on  the  floor  in 
each  case  healthy  guinea-pigs  mixed  with  guinea-pigs  artificially  infected 
with  plague  were  allowed  to  run  about  together.  In  the  first  two  sets 
of  huts  to  which  fleas  had  access  the  healthy  guinea-pigs  contracted 
plague,  while  in  the  third  set  they  remained  unaffected,  though  they 
were  freely  liable  to  contamination  by  contact  with  the  bodies  and  excreta 

1  See  Journal  of  Hygiene,  vi.  421 ;  vii.  323. 


484  PLAGUE 

of  the  diseased  animals.  In  the  third  set  of  huts  no  infection  took  place 
as  long  as  fleas  were  excluded,  but  when  accidentally  these  insects 
obtained  admission,  then  infection  of  the  uninoculated  animals  com- 
menced. Other  experiments  were  also  performed.  In  one  case  healthy 
guinea-pigs  were  suspended  in  a  cage  two  inches  above  a  floor  on  which 
infected  and  flea-infested  animals  were  running  about.  Infection  occurred 
in  the  cage,  but  if  the  latter  were  suspended  at  a  distance  above  the 
floor  higher  than  a  flea  could  jump,  then  no  infection  took  place.  Again, 
in  a  hut  in  which  guinea-pigs  had  died  of  plague,  and  which  contained 
infected  fleas,  two  cages  were  placed,  each  containing  a  monkey.  One 
cage  was  surrounded  by  a  zone  of  sticky  material  broader  than  the  jump 
of  a  flea.  The  monkey  in  this  cage  remained  unaffected,  but  the  other 
monkey  contracted  plague. 

Other  experiments  showed  that  when  plague  bacilli  were 
placed  on  the  floors  of  houses,  they  died  off  in  a  comparatively 
short  period  of  time.  After  forty-eight  hours  it  was  not  found 
possible  to  reproduce  plague  by  inoculation  with  material  from 
floors  which  had  been  grossly  contaminated  with  cultures  of  the 
bacillus.  Afterwards,  however,  animals  placed  in  such  a  house 
might  become  infected  by  means  of  fleas.  In  all  these  ex- 
periments the  common  rat-flea  of  India — Pulex  cheopis  (Roths- 
child)— was  used,  but  it  has  been  shown  that  this  flea,  when  a 
rat  is  not  available,  will  bite  a  man.  Recent  observations  show 
that  not  only  is  plague  transferable  by  means  of  fleas,  but  that 
this  is  practically  the  only  method  obtaining  in  natural  condi- 
tions, with  the  exception  that  rats  may  become  infected  by  eating 
the  carcases  of  other  animals  containing  large  numbers  of 
plague  bacilli.  It  is  improbable  from  the  experiments  made 
that  plague  is  transmitted  by  direct  contact  even  when  of  a 
close  nature;  in  fact,  it  has  been  shown  that  plague-infected 
guinea-pigs  may  suckle  their  young  without  the  latter  acquiring 
the  disease.  The  general  results  show  that  in  the  human 
subject  direct  infection  by  dust  and  other  material  through 
small  lesions  of  the  skin  plays,  probably,  a  comparatively  small 
part  in  the  spread  of  the  disease,  fleas  apparently  being  in 
nearly  all  cases  the  carriers  of  infection. 

The  more  recent  work  of  the  Committee  has  supplied  in- 
formation of  the  highest  value  with  regard  to  the  epidemiology 
of  the  disease  ;  it  has  shown,  in  short,  that  plague  in  its  epidemic 
form  is  dependent  on  the  epizootic  among  rats,  and  with  regard 
to  this  some  further  facts  may  be  given.  Plague  in  Bombay 
occurs  in  two  chief  species  of  rats,  the  mus  rattus,  the  black 
house-rat,  and  mus  decumanus,  the  grey  rat  of  the  sewers. 
The  former,  owing  to  its  presence  in  dwelling-houses,  is  chiefly 
responsible  for  the  transmission  of  the  disease  to  man  ;  while  the 
latter,  on  account  of  the  large  number  of  fleas  which  infest 


TOXINS,  IMMUNITY,  ETC.  485 

it,  is  of  special  importance  in  maintaining  the  disease  from 
season  to  season.  The  year  may  be  divided  into  two  portions 
— an  epizootic  season,  from  December  to  May  inclusive,  and  a 
non-epizootic,  from  June  to  November.  During  the  latter 
period  there  are  few  cases  of  plague  in  rats  on  account  of  fleas 
being  scanty;  especially  is  this  so  in  the  case  of  mus  rattus. 
In  fact,  in  certain  villages  where  this  species  alone  is  present, 
tin-  disease  may  actually  die  out  at  the  end  of  the  epizootic 
season,  and  accordingly  when  plague  reappears  in  these  places 
this  is  due  to  a  fresh  importation — a  fact  of  great  practical 
i 1 1 1 { M  »rtance.  A  fresh  epizootic  first  affects  chiefly  mus  decumanus, 
and  a  little  later  spreads  to  mus  rattux,  while  a  little  later  still 
the  disease  attacks  the  human  subject  in  the  epidemic  form; 
in  each  case  fleas  form  the  vehicle  of  transmission,  and  an 
interval  of  from  ten  to  fourteen  days  intervenes  between  the 
outbreak  of  the  epizootic  and  that  of  the  epidemic.  The 
proportion  of  cases  of  plague  in  mus  decumanus  is  much  higher 
than  in  /////«  rattus,  for  the  reason  mentioned.  It  has  been 
further  shown  that  the  bacilli  flourish  in  the  stomach  of  the 
flea  and  are  passed  in  a  virulent  condition  in  the  faeces,  that  a 
large  proportion  of  fleas  removed  from  plague-infected  rats 
contain  plague  bacilli,  and  that  the  fleas  may  remain  infective 
for  a  considerable  nmnlxsr  of  days,  sometimes  for  a  fortnight. 
The  repeated  contamination  of  flea-bites  by  means  of  the 
excrement  of  fleas  seems  to  be  the  most  likely  means  of  infection 
of  the  human  subject. 

hi  primary  plague  pneumonia,  from  a  consideration  of  the 
anatomical  changes  and  the  clinical  facts,  the  disease  may  be 
said  to  be  produced  by  the  direct  passage  of  the  bacilli  into  the 
respiratory  passages.  Nevertheless  there  must  be  certain  factors, 
still  imperfectly  understood,  which  determine  the  incidence  of 
this  form  :  as  in  some  epidemics  of  the  highest  virulence,  plague 
pneumonia  has  been  practically  absent,  though  opportunities  for 
infection  by  inhalation  must  have  been  present.  On  the  other 
hand,  a  case  of  plague  pneumonia  is  of  great  infectivity  in 
producing  other  cases  of  plague  pneumonia.  If  we  except 
infection  through  the  respiratory  passages  in  such  cases,  it  may 
In  said  that  direct  infection  from  patient  to  patient  is  relatively 
uncommon.  This  is  in  accordance  with  the  fact  that  in  bubonic 
plague  the  bacilli  are  not  discharged  from  the  unbroken  surface 
of  the  body,  and  are  only  present  in  the  secretions  in  severe 
cases. 

Toxins,  Immunity,  etc. — As  is  the  case  with  most  organisms 
which  extensively  invade  the  tissues,  the  toxins  in  plague 


486  PLAGUE 

cultures  are  chiefly  contained  in  the  bodies  of  the  bacteria. 
Injection  of  dead  cultures  in  animals  produces  distinctly  toxic 
effects;  post  mortem  haemorrhage  in  the  mucous  membrane 
of  the  stomach,  areas  of  necrosis  in  the  liver  and  at  the  site 
of  inoculation,  may  be  present.  The  toxic  substances  are 
comparatively  resistant  to  heat,  being  unaffected  by  an  exposure 
to  65°  C.  for  an  hour.  By  the  injection  of  dead  cultures  in 
suitable  doses,  a  certain  degree  of  immunity  against  the  living 
virulent  bacilli  is  obtained,  and,  as  first  shown  by  Yersin, 
Calmette,  and  Borrel,  the  serum  of  such  immunised  animals 
confers  a  degree  of  protection  on  small  animals  such  as  mice. 
On  these  facts  the  principles  of  preventive  inoculation  and 
serum  treatment,  presently  to  be  described,  depend.  It  may 
also  be  mentioned  that  the  filtrate  of  a  plague  culture  possesses 
a  very  slight  toxic  action,  and  the  Indian  Plague  Commission 
found  that  such  a  filtrate  has  practically  no  effect  in  the 
direction  of  conferring  immunity. 

1.  Preventive  Inoculation — Ilaffkine's  Method. — To  prepare 
the  preventive  fluid,  cultures  are  made  in  flasks  of  bouillon  with 
drops  of  oil  on  the  surface  (in  India  Haffkine  employed  a 
medium  prepared  by  digesting  goat's  flesh  with  hydrochloric 
acid  at  140°  C.  and  afterwards  neutralising  with  caustic  soda). 
In  such  cultures  stalactite  growths  (vide  supra)  form,  and  the 
flasks  are  shaken  every  few  days  so  as  to  break  up  the  stalactites 
and  induce  fresh  crops.  The  flasks  are  kept  at  a  temperature 
of  about  25°  C.,  and  growth  is  allowed  to  proceed  for  about 
six  weeks.  At  the  end  of  this  time  sterilisation  is  effected  by 
exposing  the  contents  of  the  flasks  to  65°  C.  for  an  hour ; 
thereafter  carbolic  acid  is  added  in  the  proportion  of  '5  per  cent. 
The  contents  are  well  shaken  to  diffuse  thoroughly  the  sediment 
in  the  fluid,  and  are  then  distributed  in  small  sterilised  bottles 
for  use.  The  preventive  fluid  thus  contains  both  the  dead 
bodies  of  the  bacilli  and  any  toxins  which  may  be  in  solution. 
It  is  administered  by  subcutaneous  injection,  the  dose,  which 
varies  according  to  the  "  strength,"  being  on  an  average  about 
7 '5  c.c.  Usually  only  one  injection  is  made,  sometimes  two, 
though  the  latter  procedure  does  not  appear  to  have  any 
advantage.  The  method  has  been  systematically  tested  by 
inoculating  a  certain  proportion  of  the  inhabitants  of  districts 
exposed  to  infection,  leaving  others  uninoculated,  and  then 
observing  the  proportion  of  cases  of  disease  and  the  mortality 
amongst  the  two  classes.  The  results  of  inoculation,  as  attested 
by  the  first  Indian  Commission,  have  been  distinctly  satisfactory. 
For  although  absolute  protection  is  not  afforded  by  inoculation, 


SERUM  DIAGNOSIS  487 

both  the  proportion  of  cases  of  plague  and  the  percentage 
mortality  amongst  these  cases  have  been  considerably  smaller 
in  the  inoculated,  as  compared  with  the  uninoculated.  Protec- 
tion is  not  established  till  some  days  after  inoculation,  and  lasts 
for  a  considerable  number  of  weeks,  possibly  for  several  months 
(  U;iiuu-i •man).  In  the  .Punjab  during  the  season  1902-3  the 
case  incidence  among  the  inoculated  was  1'8  per  cent.,  among 
the  uninoculated  7 '7  per  cent.,  while  the  case  mortality  was  23*9 
iiiid  60*1  per  cent,  respectively  in  the  two  classes,  the  statistics 
U'iiig  taken  from  villages  where  10  per  cent,  of  the  population 
and  upwards  had  been  inoculated. 

2.  Anti-plague  Sera. — Of  these,   two  have  been  used  as  therapeutic 
agents,  namely,  that  of  Yersin  and  that  of  Lustig.     Yersin's  serum  is 
prepared  by  injections  of  increasing  doses  of  plague  bacilli  into  the 
horse.     In  the  early  .stages  of  immunisation   dead   bacilli  are  injected 
subcutaneously,  thereafter  into  the  veins,  arid,  finally,  living  bacilli  are 
injected  intravenously.     After  a  suitable  time  blood  is  drawn  oft'  and 
the  serum  is  preserved  in  the  usual  way.     Of  this  serum  10  to  20  c.c. 
are    used,    and    injections    are    usually   repeated    on    subsequent   days. 
Lustig's  serum  is   prepared   by   injecting  a  horse   with    repeated   and 
increasing  doses  of  a  substance  derived  from  the  bodies  of  plague  bacilli, 
probably  in  great  part  nucleo-proteid.     Masses  of  growth  are  obtained 
from  the  surface  of  agar  cultures,  and  are  broken  up  and  dissolved  in  a 
1  per  cent,  solution  of  caustic  potash.     The  solution  is  then  made  slightly 
acid  by  hydrochloric  acid,   when   a  bulky   precipitate   forms  ;    this   is 
collected  on  a  filter  and  dried.     For  use  a  weighed  amount  is  dissolved 
in  a  weak  solution  of  carbonate  of  soda  and  then  injected.     The  serum 
is  obtained  from  the  animal  in  the  usual  way.     Extensive  observations 
with  both  of  these  sera  show  that  neither  of  them  can  be  considered 
a   powerful  remedy  in   cases  of  plague,    though    in   certain   instances 
distinctly    favourable  results   have  been  recorded.     The   Indian   Com- 
mis.sion,  however,  came  to  the  conclusion  "that,  on  the  whole,  a  certain 
amount   of  advantage  accrued   to  the   patients  both   in  case  of  those 
injected  with  Yersin's  serum  and  of  those  injected  with  Lustig's  serum." 
It  may  also  be  mentioned  that  the  Commission  found,  as  the  result  of 
t-xperiments,  that  Yersin's  serum  modified  favourably  the  course  of  the 
disease  in  animals,  whereas  Lustig's  serum  had  no  such  effect. 

3.  Serum  Diagnosis. — Specific  agglutinins  may  appear  in  the  blood  of 
patients  suffering  from  plague,  as  also  they  do  in  the  case  of  animals 
immunised  against  the  plague  bacillus.     It  is  to  be  noted,  however,  that 
in  clinical  cases  the  reaction  is  not  invariably  present,  the  potency  of 
the  serum  is  not  of  high  order,    and  the  carrying  out  of  the  test  is 
complicated  by  the  natural  tendency  of  the  bacilli  to  cohere  in  clumps. 
For  the  last  reason  the  macroscopic  (sedimentation)  method  is   to  be 
preferred  to  the  microscopic  (p.  120).     A  suspension  of  plague  bacilli  is 
made  by  breaking  up  a  young  agar  culture  in   "75  per  cent,    sodium 
chloride  solution  ;  the  larger  ttocculi  of  growth  are  allowed  to  settle,  and 
the  fine,  supernatant  emulsion  is  employed  in  the  usual  way.     According 
to  the  results  of  the  German  Plague  Commission  and  the  observations  of 
Cairns,  made  during  the  Glasgow  epidemic,   it  may  be  said   that  the 
reaction  is  best  obtained  with  dilutions  of  the  serum  of  from  1  :  10  to 


488  MALTA  FEVER 

1  :  50.  Cairns  found  that  the  date  of  its  appearance  is  about  a  week 
after  the  onset  of  illness,  and  that  it  usually  increases  till  about  the  end 
of  the  sixth  week,  thereafter  fading  off'.  It  is  most  marked  in  severe 
cases  characterised  by  an  early  and  favourable  crisis,  less  marked  in 
severe  cases  ultimately  proving  fatal,  whilst  in  very  mild  cases  it  is 
feeble  or  may  be  absent.  The  method,  if  carefully  applied,  may  be  of 
service  under  certain  conditions  ;  but  it  will  be  seen  that  its  use  as  a 
means  of  diagnosis  is  somewhat  restricted. 

Methods  of  Diagnosis. — Where  a  bubo  is  present  a  little  of 
the  juice  may  be  obtained  by  plunging  a  sterile  hypodermic 
needle  into  the  swelling.  The  fluid  is  then  to  be  examined 
microscopically,  and  cultures  on  agar  or  blood  serum  should  be 
made  by  the  successive  stroke  method.  The  cultural  and 
morphological  characters  are  then  to  be  investigated,  the  most 
important  being  the  involution  forms  on  salt  agar  and  the 
stalactite  growth  in  bouillon,  though  the  latter  may  not  always 
be  obtained  with  the  plague  bacillus  :  the  pathogenic  properties 
should  also  be  studied,  the  guinea-pig  being  on  the  whole  most 
suitable  for  subcutaneous  inoculation.  In  many  cases  a  diagnosis 
may  be  made  by  microscopic  examination  alone,  as  in  no  known 
condition  other  than  plague  do  bacilli  with  the  morphological 
characters  of  the  plague  bacillus  occur  in  large  numbers  in  the 
lymphatic  glands.  The  organism  may  be  obtained  in  culture 
from  the  blood  in  a  considerable  proportion  of  cases  by  with- 
drawing a  few  cubic  centimetres  and  proceeding  in  the  usual 
manner.  On  the  occurrence  of  the  first  suspected  case,  every 
care  to  exclude  possibility  of  doubt  should  be  used  before  a 
positive  opinion  is  given. 

In  a  case  of  suspected  plague  pneumonia,  in  addition  to 
microscopic  examination  of  the  sputum,  the  above  cultural 
methods  along  with  animal  inoculation  with  the  sputum  should 
be  carried  out;  subcutaneous  injection  in  the  guinea-pig  and 
smearing  the  nasal  mucous  membrane  of  the  rat  may  be  recom- 
mended. Here  a  positive  diagnosis  should  not  be  attempted  by 
microscopic  examination  alone,  especially  in  a  plague-free  dis- 
trict, as  bacilli  morphologically  resembling  the  plague  organism 
may  occur  in  the  sputum  in  other  conditions. 

MALTA  FEVER. 

Synonyms — Mediterranean  Fever:  Rock  Fever  of  Gibraltar: 
Neapolitan  Fever,  etc. 

This  disease  is  of  common  occurrence  along  the  shores  of  the 
Mediterranean  and  in  its  islands.  Since  its  bacteriology  has 


MICROCOCCUS  MELTTENSIS  489 

been  worked  out,  it  has  been  found  to  occur  also  in  India, 
China,  South  Africa,  and  in  some  parts  of  North  and  South 
America,  its  distribution  being  much  wider  than  was  formerly 
supposed.  Although  from  its  symptomatology  and  pathological 
anatomy  it  had  been  recognised  as  a  distinct  affection,  and  was 
known  under  various  names,  its  precise  etiology  was  unknown 
till  the  publication  of  the  researches  of  Colonel  Bruce  in  1887. 
From  the  spleen  of  patients  dead  of  the  disease  he  cultivated  a 
characteristic  organism,  now  known  as  the  Jficrococcus  melitensis, 
and  by  means  of  inoculation  experiments  established  its  causal 
relationship  to  the  disease.  Wright  and  Semple  applied  the 
agglutination  test  to  the  diagnosis  of  the  disease,  while  within 
recent  years  the  mode  of  spread  of  the  disease  has  been  fully 
studied  by  a  Commission,  and  it  has  been  demonstrated  that 
goat's  milk  is  the  chief  means  of  infection. 

The  duration  of  the  disease  is  usually  long — often  two  or 
three  months,  though  shorter  and  much  longer  periods  are  met 
with.  Its  course  is  very  variable,  the  fever  being  of  the  con- 
tinued type  \\ith  irregular  remissions.  In  addition  to  the  usual 
symptoms  of  pyrexia,  there  occur  profuse  perspiration,  pains 
and  sometimes  swellings  in  the  joints,  occasionally  orchitis, 
whilst  constipation  is  usually  a  marked  feature.  The  mortality 
is  low — about  2  i>er  cent.  (Bruce). 

In  fatal  cases  the  most  striking  post  mortem  change  is  in  the 
spleen.  This  organ  is  enlarged,  often  weighing  slightly  over  a 
pound,  and  in  a  condition  of  acute  congestion ;  the  pulp  is  soft 
and  may  be  diffluent,  and  the  Malpighian  bodies  are  swollen  and 
indistinct.  In  the  other  organs  the  chief  change  is  cloudy 
swelling ;  in  the  kidneys  there  may  be  in  addition  glomerular 
nephritis.  The  lymphoid  tissue  of  the  intestines  shows  none  of 
the  changes  characteristic  of  typhoid  fever. 

Micrococcus  melitensis. — This  is  a  small,  rounded,  or  slightly 
oval  organism  about  '4  /x  in  diameter,  which  is  specially  abundant 
in  the  spleen.  It  usually  occurs  singly  or  in  pairs,  but  in 
cultures  short  chains  are  also  met  with  (Fig.  147).  (Durham 
has  shown  that  in  old  cultures  kept  at  the  room  temperature 
bacillary  forms  appear,  and  we  have  noticed  indications  of  such 
in  comparatively  young  cultures :  the  usual  form  is,  however, 
that  of  a  coccus.)  It  stains  fairly  readily  with  the  ordinary 
basic  aniline  stains,  but  loses  the  stain  in  Gram's  method.  It  is 
Lr<  m-rally  said  to  be  a  non-motile  organism.  Gordon,  however, 
is  of  a  contrary  opinion,  and  has  recently  demonstrated  that  it 
possesses  from  one  to  four  nagella,  which,  however,  are  difficult 
to  stain.  In  the  spleen  of  a  patient  dead  of  the  disease  it 


490 


MALTA  FEVER 


occurs  irregularly  scattered  through  the  congested  pulp ;  it  may 
also  be  found  in  small  numbers  post  mortem  in  the  capillaries  of 
various  organs.  It  may  be  cultivated  from  the  blood  during  life 
in  a  considerable  proportion  of  cases ;  for  this  purpose  5  to  10  c.c. 
of  blood  should  be  withdrawn  from  a  vein  and  distributed  in 
small  flasks  of  bouillon.  The  micrococcus  was  found  by  the 
members  of  the  Commission  in  the  urine  of  Malta  fever  patients 
in.  10  per  cent,  of  the  cases  examined ;  it  was  sometimes  scanty, 
but  sometimes  present  in  large  numbers.  It  has  also  occasionally 
been  obtained  from  the  faeces. 

Cultivation. — This  can  usually  readily  be  effected  by  making 
stroke  cultures  on  agar  tubes  from  the  spleen  pulp  and  incub- 
ating at  37°  C.  The 
colonies,  which  are  usually 
not  visible  before  the 
third  or  fourth  day,  ap- 
pear as  small  round  discs, 
;'  l%  "  j.  slightly  raised  and  of  some- 
what transparent  appear- 
|  ance.  The  maximum  size 
— 2  to  3  mm.  in  diameter 
'"I  — is  reached  about  the 
ninth  day;  at  this  period 
by  reflected  light  they 
appear  pearly  white,  while 
by  transmitted  light  they 
have  a  yellowish  tint  in  the 
centre,  bluish  white  at  the 
periphery.  A  stroke  culture 
shows  a  layer  of  growth 
of  similar  appearance  with 

somewhat  serrated  margins.  Old  cultures  assume  a  buff  tint. 
The  optimum  temperature  is  37°  C.,  but  growth  still  occurs  down 
to  about  20°  C.  On  gelatin  at  summer  temperature  growth  is 
extremely  slow — after  two  or  three  weeks,  in  a  puncture  culture, 
there  is  a  delicate  line  of  growth  along  the  needle  track  and  a 
small  flat  expansion  of  growth  on  the  surface.  There  is  no 
liquefaction  of  the  medium.  In  bouillon  there  occurs  a  general 
turbidity  with  flocculent  deposit  at  the  bottom  ;  on  the  surface 
there  is  no  formation  of  a  pellicle.  The  reaction  of  the  media 
ought  to  be  very  faintly  alkaline,  as  marked  alkalinity  interferes 
with  the  growth;  a  reaction  of  +  10  (p.  34)  has  been  found 
very  suitable.  On  potatoes  no  visible  growth  takes  place  even 
at  the  body  temperature,  though  the  organism  multiplies  to  a 


FIG.  147. — Micrococcus  meliteusis,  from  a 

two  days'  culture  on  agar  at  37°  C. 

Stained  with  fuchsin.      x  1000. 


MODE  OF  SPREAD  OF  THE  DISEASE          491 

certain  extent.  Outside  the  body  the  organism  has  considerable 
[•owns  of  vitality,  as  it  has  been  found  to  survive  in  a  dry  con- 
dition in  dust  and  clothing  for  a  period  of  two  months. 

Relations  to  the  Disease. — There  is  in  the  first  place  ample 
evidence,  from  examination  of  the  spleen,  both  post  mortem  and 
during  life,  that  this  organism  is  always  present  in  the  disease. 
The  exj>eriments  of  Bruce  and  Hughes  first  showed  that  by 
inoculation  with  even  comparatively  small  doses  of  pure  cultures 
the  disease  could  be  produced  in  monkeys,  sometimes  with  a 
fatal  result.  And  it  has  now  been  fully  established  that  inocula- 
tion with  the  minutest  amount  of  culture,  even  by  scarification, 
leads  to  infection  both  in  monkeys  and  in  the  human  subject. 

liabbits,  guinea-pigs,  and  mice  are  insusceptible  to  inoculation 
by  the  ordinary  method.  Durham,  by  using  the  intracerebral 
method  of  inoculation,  however,  succeeded  in  raising  the  virul- 
ence, so  that  the  organism  is  capable  of  producing  in  guinea- 
pigs  on  intraperitoneal  injection  illness  with  sometimes  a  fatal 
result  many  weeks  afterwards.  An  interesting  point  brought 
out  by  these  experiments  is  that,  in  the  case  of  animals  which 
survive,  the  micrococcus  may  be  cultivated  from  the  urine  several 
months  after  inoculation. 

Mode  of  Spread  of  the  Disease. — The  work  of  the  recent 
Commission  has  resulted  in  establishing  facts  of  the  highest 
importance  with  regard  to  the  spread  of  the  disease.  In  the 
course  of  investigations  Zammitt  found  that  the  blood  of  many 
of  the  goats  agglutinated  the  micrococcus  melitensis,  and 
Horrocks  obtained  cultures  of  the  organism  from  the  milk. 
Further  observations  showed  that  agglutination  was  given  in 
the  case  of  50  per  cent,  of  the  goats  in  Malta,  whilst  the  organism 
was  present  in  the  milk  in  10  per  cent.  Sometimes  the  organism 
was  present  in  enormous  numbers,  and  in  these  cases  the  animal 
usually  appeared  poorly  nourished,  whilst  the  milk  had  a  some- 
what serous  character.  In  other  cases,  however,  the  organism 
was  found  when  the  animals  appeared  healthy,  and  there  was 
no  physical  or  chemical  change  in  the  milk.  It  was  also 
determined  that  the  organism  might  be  excreted  for  a  period 
of  two  to  three  months  before  any  notable  change  occurred  in 
the  milk.  Agglutination  is  usually  given  by  the  milk  of  infected 
animals,  and  this  property  was  always  present  when  the  micro- 
coccus  was  found  in  the  milk.  It  was,  moreover,,  found  that 
monkeys  and  goats  could  be  readily  infected  by  feeding  them 
with  milk  containing  the  micrococcus,  the  disease  being  contracted 
by  fully  80  per  cent,  of  the  monkeys  used.  It  was  therefore 
rendered  practically  certain  that  the  human  subject  was  infected 


492  MALTA  FEVER 

by  means  of  such  milk,  and  the  result  of  preventive  measures 
by  which  milk  was  excluded  as  an  article  of  dietary  amongst 
the  troops  in  Malta  has  fully  borne  out  this  view.  After  such 
measures  were  instituted,  the  number  of  cases  in  the  second 
half  of  1906  fell  to  11  per  thousand,  as  contrasted  with  47 
per  thousand  in  the  corresponding  part  of  the  preceding  year ; 
further  successful  results  have  followed.  The  various  facts  with 
regard  to  the  epidemiology  of  the  disease  have  thus  been  cleared 
up.  For  example,  it  is  more  prevalent  in  the  summer  months, 
when  more  milk  is  consumed,  and  there  is  a  larger  proportion 
of  cases  amongst  those  in  good  social  position,  the  officers,  for 
example,  suffering  more  in  proportion  than  the  privates.  Another 
interesting  fact,  pointed  out  by  Horrocks,  is  that  the  disease  has 
practically  disappeared  from  .Gibraltar  since  the  practice  of  im- 
porting goats  from  Malta  has  stopped. 

The  work  of  the  Commission,  so  far  as  it  has  gone,  has  been 
to  exclude  other  modes  of  infection  than  the  ingestion  of  infected 
milk  as  being  of  practical  importance ;  if  the  disease  is  conveyed 
by  contact  at  all,  this  is  only  when  the  contact  is  of  an  intimate 
character,  and  even  then  it  is  probably  of  rare  occurrence.  Al- 
though numerous  patients  suffering  from  the  disease  come  to 
England,  there  is  no  known  case  of  fresh  infection  arising  under 
natural  conditions. 

There  is  distinct  evidence  that  the  disease  may  be  acquired  by 
inoculation  through  small  lesions  in  the  skin,  and  this  method 
is  probably  not  infrequent  amongst  those  who  handle  infected 
milk.  It  has  been  shown  that  the  organism  may  remain  alive 
in  the  bodies  of  mosquitoes  for  four  or  five  days,  and  possibly 
these  insects  may  occasionally  be  the  means  of  carrying  the 
disease ;  there  is  no  evidence,  however,  that  this  takes  place 
to  any  extent. 

Agglutinative  Action  of  Serum. — The  blood  serum  of  patients 
suffering  from  Malta  fever  possesses  the  power  of  agglutinating 
the  micrococcus  melitensis  in  a  manner  analogous  to  what  has 
been  described  in  the  case  of  typhoid  fever.  The  reaction 
appears  comparatively  early,  often  about  the  fifth  day,  and  may 
be  present  for  a  considerable  time  after  recovery — sometimes 
for  more  than  a  year.  Distinct  agglutination  with  a  1  :  30 
dilution  of  the  serum  in  half  an  hour  may  be  taken  as  a  positive 
reaction,  sufficient  for  diagnosis.  The  reaction  is,  however, 
usually  given  by  much  higher  dilutions,  e.g.  1  : 500,  and  even 
higher.  It  is  to  be  noted  that  normal  serum  diluted  1  :  5  may 
produce  some  agglutination.  As  regards  relation  to  prognosis, 
the  observations  of  Birt  and  Lamb  and  of  Bassett-Smith 


METHODS  OF  DIAGNOSIS  493 

have  given  results  analogous  to  those  obtained  in  typhoid 
(p.  372). 

The  Commission  has  found  that  vaccination  with  dead  cultures 
of  the  micrococcus  confers  a  certain  degree  of  protection  amongst 
those  exposed  to  the  disease.  As  a  rule  two  injections  were 
made,  200-300  million  cocci  being  the  dose  of  the  first  injection, 
and  about  400  million  the  dose  of  the  second.  The  use  of  vaccines 
has  also  been  carried  out  in  the  treatment  of  the  disease,  but 
the  observations  are  not  sufficiently  numerous  to  allow  a  definite 
Muit'iiM'iit  to  be  made  as  to  its  value. 

Methods  of  Diagnosis. — During  life  the  readiest  means  of 
diagnosis  is  supplied  by  the  agglutinative  test  just  described 
(for  technique,  vide  p.  118). 

Cultures  are  most  easily  obtained  from  the  spleen  either 
during  life  or  post  mortem.  Inoculate  a  number  of  agar  tubes 
by  successive  strokes  and  incubate  at  37°  C.  Film  preparations 
should  also  be  made  from  the  spleen  pulp  and  stained  with 
carbol-thionin-blue  or  diluted  carbol-fuchsin  (1  :  10).  Cultures 
may  sometimes  be  obtained  from  the  blood  by  the  usual 
methods. 


CHAPTER   XX. 

DISEASES  DUE  TO  SPIROCHAETES— THE  RELAPSING 
FEVERS,  SYPHILIS,  AND  FRAMBCESIA. 

THE  diseases  produced  by  spirochaetes — spirilloses  or  spiro- 
chaetoses — fall  into  two  main  groups,  one  represented  by  the 
human  spirillar  fevers  and  the  corresponding  affections  of  various 
animals,  and  the  second  having  as  its  two  chief  members 
syphilis  and  yaws,  though  to  the  organisms  of  these  diseases 
various  spirochaetes  found  in  ulcerative  and  gangrenous  con- 
ditions seem  to  be  closely  related.  The  members  of  the  first 
group  are  essentially  blood  infections,  and  the  organisms  are  in 
most,  if  not  in  all  cases,  transmitted  by  blood-sucking  ecto- 
parasites ;  in  the  second  group  the  organisms  are  primarily 
tissue-parasites,  blood  infection  when  it  occurs  being  a  later 
phenomenon,  and  infection  would  appear  to  occur  by  direct 
contact.  As  regards  general  morphology,  staining  reactions, 
conditions  of  growth  and  culture,  the  various  spirochaetes 
present  certain  common  characters,  and,  as  already  stated,  it 
is  still  uncertain  whether  they  are  to  be  regarded  as  bacteria 
or  as  protozoa,  though  the  balance  of  opinion  is  now  distinctly 
in  favour  of  the  latter. 

RELAPSING  FEVER  AND  AFRICAN  TICK  FEVER. 

At  a  comparatively  early  date,  namely  in  1873,  when  prac- 
tically nothing  was  known  with  regard  to  the  production  of 
disease  by  bacteria,  a  highly  characteristic  organism  was  dis- 
covered by  Obermeier  in  the  blood  of  patients  suffering  from 
relapsing  fever.  This  organism  is  usually  known  as  the 
spirillum  or  spirochcete  Obermeieri,  or  the  spirillum  of  relapsing 
fever.  He  described  its  microscopical  characters,  and  found 
that  its  presence  in  the  blood  had.  a  definite  relation  to  the 
time  of  the  fever,  as  the  organism  rapidly  disappeared  about 
the  time  of  the  crisis,  and  reappeared  when  a  relapse  occurred. 

41)4 


THE  SPIROCH^ETE  OBERMETERI  495 

His  observations  were  fully  confirmed,  and  his  views  as  to  its 
causal  relationship  to  the  disease  have  been  established  as 
correct. 

Within  recent  years  relapsing  fever  has  been  carefully  studied 
in  different  parts  of  the  world,  and  the  relationships  of  the 
organisms  have  been  the  subject  of  much  investigation  and 
discussion.  This  question  will  be  referred  to  again  below. 
Recently  also  it  has  been  shown  that  the  so-called  "  tick  fever  " 
prevalent  in  Africa  is  due  to  a  spirochsete  of  closely  similar 
character,  and  results  of  the  highest  importance  have  been 
established  with  regard  to  the  part  played  by  ticks  in  the 
transmission  of  the  disease.  As  a  matter  of  convenience,  we 
shall  give  the  chief  facts  regarding  these  diseases  separately. 
It  has  also  been  shown  that  spirillar  diseases  or  "  spirilloses," 
as  they  are  called,  are  widespread  amongst  vertebrates;  they 
have  been  described,  for  example,  in  geese  by  Sacharoff,  in 
fowls  by  Marchoux  and  Salimbeni,  in  oxen  and  sheep  by 
Theiler,  and  in  bats  by  Nicolle  and  Comte,  and  it  is  interesting 
to  note  that  in  the  case  of  the  spirilloses  of  oxen  and  fowls  the 
infection  is  transmissible  by  means  of  ticks. 

Characters  of  the  Spirochaete. — The  organisms  as  seen  in 
the  blood  during  the  fever  are  delicate  spiral  filaments  which 
have  a  length  of  from  two  to  six  times  the  diameter  of  a  red  blood 
corpuscle.  They  are,  however,  exceedingly  thin,  their  thickness 
being  much  less  than  that  of  the  cholera  spirillum.  They  show 
several  regular  sharp  curves  or  windings,  of  number  varying 
according  to  the  length  of  the  organisms,  and  their  extremities  are 
finely  pointed  (Fig.  148).  There  are  often  to  be  seen  in  the 
spirals,  portions  which  are  thinner  and  less  deeply  stained  than 
tlio  rest,  and  which  suggest  the  occurrence  of  transverse  division. 
They  are  actively  motile,  and  may  be  seen  moving  quickly 
across  the  microscopic  field  with  a  peculiar  movement  which 
is  partly  twisting  and  partly  undulatory,  and  disturbing  the 
blood  corpuscles  in  their  course. 

They  stain  with  watery  solutions  of  the  basic  aniline  dyes, 
though  somewhat  faintly,  and  are  best  coloured  by  the 
Romanowsky  method  or  one  of  its  modifications.  When  thus 
stained  they  usually  have  a  uniform  appearance  throughout,  or 
may  be  slightly  granular  at  places,  but  they  show  no  division 
into  short  segments.  They  lose  the  stain  in  Gram's  method. 
There  is  no  evidence  that  they  form  spores. 

Novy  found  that  the  spirochaete  of  American  relapsing  fever 
remained  alive  and  virulent  in  defibrinated  rats'  blood  for 
forty  days.  He  also  succeeded,  by  Levaditi's  method  (p.  503), 


496 


RELAPSING  FEVER 


in  obtaining  cultures  in  collodion  sacs  containing  rats'  blood 
which  were  placed  in  the  peritoneum  of  rats.  By  this  method 
cultures  were  maintained  for  many  generations ;  the  organisms 
were  still  virulent  though  the  resulting  infection  was  rather  less 
intense  than  at  first.  The  spirochsetes  are  readily  killed  at  a 
temperature  of  60°  C.,  but  may  be  exposed  to  0°  C.  without 
being  killed.  Novy  and  Knapp  have  found  that  there  is  a  single 
flagellum  at  one  end  of  this  organism. 

Relations  to  the  Disease. — -In  relapsing  fever,  after  a  period 
of  incubation  there  occurs  a  rapid  rise  of  temperature  which 
lasts  for  about  five  to  seven  days.  At  the  end  of  this  time  a 

crisis  occurs,  the  tempera- 
ture falling  quickly  to 
normal.  In  the  course 
of  about  other  seven 
days  a  sharp  rise  of 
temperature  again  takes 
place,  but  on  this  occa- 
sion the  fever  lasts  a 
shorter  time,  again  sud- 
denly disappearing.  A 
second  or  even  third 
relapse  may  occur  after 
a  similar  interval.  The 
organisms  begin  to  appear 
in  the  blood  shortly 
before  the  onset  of  the 
pyrexia,  and  during  the 
rise  of  temperature  rapid- 
ly increase  in  number. 
They  are  very  numerous 

during  the  fever,  a  large  number  being  often  present  in 
every  field  of  the  microscope  when  the  blood  is  examined  at 
this  stage.  They  begin  to  disappear  shortly  before  the  crisis : 
after  the  crisis  they  are  entirely  absent  from  the  circulating 
blood.  A  similar  relation  between  the  presence  of  the  organ- 
isms in  the  blood  and  the  fever  is  found  in  the  case  of  the 
relapses.  Munch  in  1876  produced  the  disease  in  the  human 
subject  by  injecting  blood  containing  the  spirochsetes,  and  this 
experiment  has  been  several  times  repeated  with  the  same  result. 
Additional  proof  that  the  organism  is  the  cause  of  the  disease 
has  been  afforded  by  experiments  on  animals.  Carter  in  1879 
was  the  first  to  show  that  the  disease  could  be  readily  produced  in 
monkeys,  and  his  experiments  were  confirmed  by  Koch.  In  such 


FIG.  148.  —  Spiroohretes  of  relapsing  fever  in 
human  blood.  Film  preparation.  (After 
Koch.)  See  also  Plate  IV.,  Fig.  18. 
x  about  1000. 


IMMUNITY  497 

experiments  the  blood  taken  from  patients  and  containing  the 
spirochaetes  was  injected  subcutaneously.  In  the  disease  thus 
produced  there  is  an  incubation  period  which  usually  lasts  about 
three  days.  At  the  end  of  that  time  the  organisms  rapidly  appear 
in  the  blood,  and  shortly  afterwards  the  temperature  quickly 
rises.  The  period  of  pyrexia  usually  lasts  for  two  or  three  days, 
and  is  followed  by  a  marked  crisis.  As  a  rule  there  is  no  relapse, 
but  occasionally  one  of  short  duration  occurs.1  White  mice  and 


1  •'!«..  119.—  Spirochsete  Obernieieri  in  blood  of  infected  mouse. 
xlOOO. 

rats  are  also  susceptible  to  infection.  In  the  former  animals 
the  disease  is  characterised  by  several  relapses,  in  the  latter  there 
is,  however,  no  relapse. 

Immunity.— Metclmikoff  found  that  during  the  fever  the 
spirocluL-tes  were  practically  never  taken  up  by  the  leucocytes  in 
the  circulating  blood,  but  that  at  the  time  of  the  crisis,  on  dis- 
;ijipi'jiring  from  the  blood,  they  accumulated  in  the  spleen  and 
were  ingested  in  large  numbers  by  the  microphages  or  poly- 
Morris,  Pappeuheimer,  and  Flournoy,  in  their  experiments  on  monkeys 
with  the  organism  of  American  relapsing  fever,  found  that  several  relapses 
occurred. 


498  RELAPSING  FEVER 

morphonuclear  leucocytes.  Within  these  they  rapidly  under- 
went degeneration  and  disappeared.  It  is  to  be  noted  in  this 
connection  that  swelling  of  the  spleen  is  a  very  marked  feature 
in  relapsing  fever.  These  observations  were  confirmed  by 
Soudakewitch,  who  also  found  that  when  the  disease  was  pro- 
duced in  splenectomised  monkeys  (cercocebus  fuliyinosus)  the 
spirochaetes  did  not  disappear  from  the  blood  at  the  usual  time, 
but  rather  increased  in  number,  and  a  fatal  result  followed  on 
the  eighth  and  ninth  days  respectively.  Recent  observations, 
however,  indicate  that,  as  in  the  case  of  so  many  other  diseases, 
the  all-important  factor  in  the  destruction  of  the  organisms  is 
the  development  of  antagonistic  substances  in  the  blood.  Lamb 
found  in  the  case  of  the  monkey  (macacus  radiatus)  that  the 
removal  of  the  spleen  of  an  animal  rendered  immune  by  an 
attack  of  the  disease  did  not  render  it  susceptible  to  fresh 
inoculation,  and  attributed  the  immunity  to  the  presence  of 
bactericidal  bodies  in  the  serum.  He  found,  for  example,  that 
in  vitro  the  serum  of  an  immune  animal  brought  the  movements 
of  the  spirilla  to  an  end,  clumped  them,  and  caused  their  dis- 
integration; and  further,  that  when  the  spirochaetes  and  the 
immune  serum  were  injected  in  one  case  into  a  fresh  monkey  no 
disease  developed.  In  opposition  to  Soudakewitch,  Lamb  found 
that  with  a  monkey  from  which  the  spleen  had  been  removed 
death  did  not  occur  after  it  was  inoculated  with  the  spirochaetes. 
Observations  by  Sawtschenko  and  Milkich,  Novy  and  Knapp, 
and  others,  also  show  that  in  the  course  of  infection  there  are 
developed  anti-substances  of  the  nature  of  immune-bodies,  with 
protective  properties,  and  agglutinins.  Novy  and  Knapp  pro- 
duced a  "hyper-immunity  "  in  rats  by  repeated  injections  of  blood 
containing  the  spirochaetes,  and  found  that  the  serum  of  such 
animals  had  a  markedly  curative  effect,  and  could  cut  short  the 
disease  in  rats,  mice,  and  monkeys.  The  course  of  events  in  the 
human  disease  might  be  explained  by  supposing  that  immunity 
of  short  duration  is  produced  during  the  first  period  of  pyrexia, 
but  that  it  does  not  last  until  all  the  organisms  have  been 
destroyed,  some  still  surviving  in  internal  organs  or  in  tissues 
where  they  escape  the  bactericidal  action  of  the  serum.  With 
the  disappearance  of  the  immunity  the  organisms  appear  in  the 
blood,  the  relapse  being,  however,  of  shorter  duration  and  less 
severe  than  the  first  attack.  This  is  repeated  till  the  immunity 
lasts  long  enough  to  allow  all  the  organisms  to  be  killed. 

Varieties. — As  already  stated,  relapsing  fever  has  been  studied 
in  different  parts  of  the  world,  and,  apart  from  the  African  tick 
fever,  European,  Asiatic,  and  American  types  have  been  dis- 


VARIETIES  499 

tinguished.  Differences  have  been  made  out  with  regard  to 
clinical  features,  pathogenic  effects,  and  immunity  reactions. 
Of  these  the  last  mentioned  are  probably  the  most  important. 
It  has  been  shown,  for  example,  by  the  work  of  Novy,  Strong, 
and  Mackie,  that  the  American  spirochaete  is  probably  a  distinct 
species,  as  animals  immunised  against  it  are  still  susceptible  to 
infection  by  the  European  and  Asiatic  organisms,  and  vice  versa. 
The  relationship  between  the  two  latter  is  certainly  closer,  and 
no  distinct  immunity  differences  have  been  established.  Re- 
lapsing fever  in  Asia  is  evidently  a  much  more  severe  disease 
than  in  Europe ;  Mackie  gives  the  mortality  in  Bombay  at  the 
comparatively  high  figure  of  38  per  cent.  But  differences  in  this 
respect,  as  well  as  in  pathogenic  effects,  may  simply  depend  on 
variations  in  virulence.  At  present  no  definite  statement  can  be 
made  on  this  point. 

The  fact  that  tick  fever  and  other  spirilloses  are  con- 
veyed by  the  bites  of  insects  makes  it  extremely  probable  that 
relapsing  fever  is  transmitted  in  this  way.  At  first  the  bed-bug 
was  believed  to  be  the  vehicle  of  transmission,  and  the  experi- 
ments of  Karlinski  and  of  Tictin,  which  showed  that  the  spiro- 
clia-tes  might  remain  alive  and  virulent  in  the  body  of  this 
insect  for  some  time  after  it  had  sucked  the  blood  of  a  patient, 
lent  some  support  to  this  view.  Attempts  to  transmit  the 
disease  by  means  of  the  bites  of  bugs  were,  however,  generally 
unsuccessful ;  Mackie  produced  the  disease  in  only  one  out  of 
six  monkeys  used  for  this  purpose,  though  large  numbers  of  bugs, 
which  had  bitten  relapsing  fever  patients,  were  used.  On  in- 
vestigating an  epidemic  of  the  disease,  however,  he  obtained 
a  considerable  amount  of  evidence  on  epidemiological  grounds 
that  the  disease  was  carried  by  the  body  louse.  He  also  found 
that  the  spirochaites  in  the  blood  which  had  been  sucked  under- 
went great  multiplication  about  three  days  afterwards,  and 
formed  large  tangled  masses  in  the  stomach  contents.  The 
view  that  the  louse  is  the  agent  of  transmission  of  the  human 
disease  is  strongly  supported  by  the  experiments  of  Manteufel, 
who  was  able  to  transmit  infection  from  rat  to  rat  in  nearly 
60  per  cent,  of  the  experiments  made,  whereas  he  obtained 
only  negative  results  by  means  of  bugs.  Further  observations 
are  still  necessary. 

African  Tick  Fever. 

The  disease  long  known  by  this  name  as  prevalent  in  Africa 
has  also  been  shown  to  be  caused  by  a  spirillum  or  spirocha •!<•. 


500  AFRICAN  TICK  FEVER 

Sp.  Duttoni.  Organisms  of  this  nature  had  been  seen  in  the  blood 
of  patients  in  Uganda  by  Greig  and  Nabarro  in  1903,  and  Milne 
and  Ross  in  the  end  of  1904  recorded  a  series  of  observations 
which  led  them  to  the  conclusion  that  tick  fever  was  due  to  a 
spirochyete.  It  is,  however,  chiefly  owing  to  the  work  of  Button 
and  Todd  in  the  Congo  Free  State,  on  the  one  hand,  and  of  Koch 
in  German  East  Africa,  on  the  other,  that  our  knowledge  of  this 
disease  has  been  thoroughly  established. 


FIG.  150. — Film  of  human  blood  containing  spiroehiete  of  tick  fever, 
x  lOOO.i 

The  following  are  the  chief  facts  regarding  this  fever : 
Clinically  the  fever  closely  resembles  relapsing  fever,  but  the 
periods  of  fever  are  somewhat  shorter,  rarely  lasting  for  more 
than  two  or  three  days.  It  is  seldom  attended  with  a  fatal  result 
unless  in  patients  debilitated  by  other  causes.  The  organisms 
are  considerably  fewer  in  the  blood  than  in  the  European  re- 
lapsing fever,  and  sometimes  a  careful  search  may  be  necessary 
before  they  are  found.  Morphologically,  they  are  said  to  be 

1"We  are  indebted  to  Lieut. -Col.  Sir  William  Leishman,  R.A.M.C., 
for  the  preparations  from  which  Figs.  149-151  were  taken. 


ATKLCAN  TICK  FEVER  501 

practically  identical,  although  Koch  thought  that  the  organisms 
in  tick  fever  tended  on  the  whole  to  be  slightly  longer;  the 
average  length  may  )>e  said  to  be  15  to  35  JJL.  Button  and  Todd 
showed  that  it  was  possible  to  transmit  the  disease  to  certain 
monkeys  (cercopitheci)  by  means  of  ticks  which  had  been  allowed 
to  bite  patients  suffering  from  the  disease,  the  symptoms  in 
these  animals  appearing  about  five  days  after  inoculation.  The 
disease  thus  produced  is  characterised  by  several  relapses,  and 


o    •  -    •  - 


FK..  151. — Spirillum  of  human  tick  fever  (Spirillum  Duttoni)  in 
blood  of  infected  mouse,      x  1000. 


often  leads  to  a  fatal  result.  In  one  case  they  produced  the 
disease  by  means  of  young  ticks  hatched  from  the  eggs  of  ticks 
which  had  been  allowed  to  suck  the  blood  of  fever  patients,  and 
they  came  to  the  conclusion  that  the  spirochaetes  were  not  simply 
carried  mechanically  by  the  ticks,  but  probably  underwent  some 
cycle  of  development  in  the  tissues  of  the  latter.  The  species  of 
tick  concerned  is  the  onutkodonu  moubata.  These  results  were 
confirmed  and  extended  by  Koch.  He  found  that  after  the  ticks 
had  been  allowed  to  suck  the  blood  containing  the  organisms, 
could  be  found  for  a  da  or  two  \n  the  stomachs  of  ^he 


502  AFRICAN  TICK  FEVER 

insects.  After  this  time  they  gradually  disappeared  from  the 
stomach,  but  were  detected  in  large  numbers  in  the  ovaries  of 
the  female  ticks,  where  they  sometimes  formed  felted  masses. 
He  also  traced  the  presence  of  the  spirochsetes  in  the  eggs  laid  by 
the  infected  ticks,  and  in  the  young  embryos  hatched  from  them. 
On  the  other  hand,  Leishman  has  failed  to  find  any  evidence  of 
spirochsetes  in  the  tissues  of  ticks  later  than  ten  days  after 
ingestion  of  blood  containing  them,  or  in  the  ova  laid  by  them, 
or  in  the  young  ticks  when  hatched,  though  these  were  proved 
by  experiment  to  be  infective.  After  ingestion  of  the  blood  by 
the.  ticks,  he  found  that  morphological  changes  occurred  in  the 
spirochsetes,  resulting  in  the  formation  of  minute  chromatin 
granules  which  traverse  the  walls  of  the  intestine  and  are  taken 
up  by  the  cells  of  the  Malpighian  tubules ;  they  also  penetrate 
the  ovaries  and  may  be  found  in  large  numbers  within  the  ova. 
Similar  granules  are  to.be  seen  in  the  Malpighian  tubules  of  the 
embryo  ticks,  where  they  are  also  found  in  the  subsequent  stages 
of  their  life.  He  has  abundantly  proved  that  infection  of 
animals  may  be  produced  by  inoculation  with  crushed  material 
containing  the  granules  but  no  spirocluvtes.  He  accordingly 
considers  that  the  granules  in  question  probably  represent  a 
phase  in  the  life  history  of  the  parasite,  and  that  infection  probably 
occurs  by  inoculation  of  the  skin  with  the  chromatin  granules 
voided  in  the  Malpighian  secretion  and  not  by  unaltered 
spirochsetes  from  the  salivary  glands.  It  is  also  interesting  to 
note  that  Balfour  has  found  similar  granules  in  ticks  (argas 
persicus)  infected  with  tpirochaste  gallinarwn, 

Koch  also  made  extensive  observations  on  the  ticks  in  Ger- 
man East  Africa,  and  found  that  of  over  six  hundred  examined 
11  per  cent,  of  these  insects  along  the  main  caravan  routes  con- 
tained spirilla,  and  in  some  localities  almost  half  of  the  ticks 
were  infected.  In  places  removed  from  the  main  lines  of  com- 
merce he  still  found  them,  though  in  smaller  number.  It  has 
also  been  demonstrated  that  in  some  places  the  ticks  are  found 
to  be  infected  with  the  spirilla  although  the  inhabitants  do  not 
suffer  from  tick  fever,  a  circumstance  which  is  probably  due  to 
an  acquired  immunity  against  the  disease. 

It  is  now  generally  considered  that  the  sp.  Duttoni  is  a 
species  distinct  from,  though  closely  allied  to,  the  organisms  of 
the  relapsing  fevers  described  above.  We  have  mentioned  some 
differences  in  the  clinical  characters  of  the  diseases,  and  there 
are  also  differences  in  the  pathogenic  effects  of  the  organisms  on 
inoculation.  The  sp.  Duttoni,  for  example,  produces  a  much 
more  severe  disease  in  monkeys,  and  is  pathogenic  to  more 


SYPHILIS  503 

«4>ecies  of  the  laboratory  animals  than  the  sp.  Obermeieri.  The 
most  important  differences  are  however  brought  out  by  immunity 
reactions.  It  was  shown  by  Breinl  that  the  immunity  produced 
by  the  sp.  Obermeieri  did  not  protect  against  the  sp.  Duttoni, 
and  that  the  converse  also  held  good ;  and  it  has  since  been 
established  that  a  similar  difference  obtains  between  the  sp. 
Duttoni  and  the  organisms  of  the  Asiatic  and  American  varieties 
of  relapsing  fever.  Corresponding  results  are  obtained  on 
testing  the  various  serum  reactions  in  vitro. 

Levaditi  has  succeeded  in  obtaining  cultures  of  the  spirochsete 
of  tick  fever  by  inoculating  sacs  filled  with  monkey's  serum, 
heated  at  70°  C.,  and  placing  the  sacs  in  the  peritoneal  cavity  of 
a  rat  or  rabbit ;  when  opened  at  the  end  of  five  to  seven  days, 
the  sacs  were  found  to  contain  an  abundant  growth  of  spiro- 
chaetes,  some  of  which  were  of  unusually  great  length.  Growth 
was  maintained  in  similar  sub-cultures,  and  the  virulence  was 
well  preserved. 

SYPHILIS, 

Up  till  quite  recent  times  practically  nothing  of  a  definite 
nature  was  known  regarding  the  etiology  of  syphilis.  Most 
interest  for  a  long  time  centred  around  the  observations  of 
Lustgarten,  who  in  1884  described  a  characteristic  bacillus, 
both  in  the  primary  sore  and  in  the  lesions  in  internal  organs. 
This  organism  occurred  in  the  form  of  slender  rods,  straight,  or 
slightly  bent,  3  to  4  /u,  in  length,  often  forming  little  clusters 
either  within  cells  or  lying  free  in  the  lymphatic  spaces ;  it  took 
up  basic  aniline  dyes  with  some  difficulty,  but  was  much  more 
easily  decolorised  by  acids  than  the  tubercle  bacillus.  The 
etiological  relationship  of  the  organism  to  the  disease  was, 
however,  not  generally  accepted,  and  in  view  of  the  recent  work 
on  syphilis,  the  organism  cannot  be  regarded  as  having  any 
pathological  importance. 

Spirochaete  pallida. — An  entirely  new  light  has  been  thrown 
on  the  etiology  of  the  disease  by  the  work  of  Schaudinn  and 
Hoffmann  which  appeared  in  1905.  Since  their  first  publication 
a  great  amount  of  work  has  been  undertaken  in  order  to  test 
their  conclusions,  and  the  results  have  been  of  a  confirmatory 
nature.  These  observers  found  in  cases  of  syphilis  an  organism 
to  which  they  gave  the  name  spirochcete  pallida ;  it  now  also 
goes  by  the  name  spironema  pallidum.  As  described  by  them, 
it  is  a  minute  spiral-shaped  organism,  showing  usually  from  six 
to  eight  curves,  though  longer  forms  are  met  with ;  the  curves 


504  SYPHILIS 

are  small,  comparatively  sharp,  and  regular  (Figs.  152,  153).  It 
may  be  said  to  measure  4  to  1 4  /A  in  length,  while  it  is  extremely 
thin,  its  thickness  being  only  '25  /x.  In  a  fresh  specimen,  say  a 
scraping  from  a  chancre  suspended  in  a  little  salt  solution,  the 
organism  shows  active  movements,  which  are  of  three  kinds — 
rotation  about  the  long  axis,  gliding  movements  to  and  fro, 
and  movements  of  flexion  of  the  whole  body.  The  ends  are 
pointed  and  tapering.  Its  detection  is  comparatively  difficult, 
as  the  organism  is  feebly  refractile,  and  more  difficult  to  see  than 
most  other  organisms ;  the  movement  of  small  particles  in  the 
vicinity,  however,  is  of  asistance  in  finding  it.  The  use  of  the 


FIGS.  152  and  153. — Film  preparations  from  juice  of  hard  chancre 
showing  spirochaete  pallida, — Giemsa's  stain.  xlOOO.  (From  pre- 
parations by  Dr.  A.  MacLennan.) 

parabolic    sub-stage    condenser   (p.  93)    is   of   great  service   in 
searching  for  the  organism. 

In  ulcerated  syphilitic  lesions  other  organisms  are,  of  course, 
present,  and  not  infrequently  another  spiral  organism,  to  which 
the  name  spirochaete  refringens  has  been  given.  This  organism 
is  usually  somewhat  longer,  and  is  distinctly  thicker  than  the 
spirochsete  pallida.  As  the  name  implies,  it  is  more  highly 
refractile,  and  it  is  much  more  easily  detected  than  the  latter 
organism;  its  curves  also  are  more  open  and  much,  less  regular, 
and  they  vary  in  their  appearance  during  the  movements.  In 
stained  films  (see  p.  115),  the  differences  between  the  organisms 
come  out  more  distinctly,  as  can  be  gathered  from  the  accom- 
panying photograph  (Fig.  156).  The  spirochaete  pallida  by  the 
Giemsa  stain  is  coloured  somewhat  faintly,  and  of  reddish  tint, 
whilst  the  regular  spiral  twistings  are  preserved ;  the  spirochsete 
refringens  shows  flatter,  wave-like  bends,  and,  like  other  organ- 
isms, is  stained  of  a  bluish  tint.  By  using  Loffler's  stain  for  the 


SPIROCH^ETE  PALLTDA  505 

flagella  <>f  bacteria,  Schaudinn  was  able  to  demonstrate  a  single 
delicate  fiagelluui  at  each  pole  of  the  spirochajte  pallida,  while 
no  undulating  membrane  could  be  detected;  on  the  other  hand, 
several  other  species,  including  the  spirochyete  refringens,  showed 
a  distinct  undulating  membrane.  Two  fiagella  at  one  pole  of 
the  spirorlui'te  pallida  were  also  seen,  an  appearance  which 
Schaudinn  thought  might  represent  the  commencement  of 
longitudinal  fission. 

The  number  of  publications  with  regard  to  the  distribution  of 
the  spiroclut'tf  pallida  is  already  very  large,  and  a  summary  of 


FIG.  ].">!.  Film  preparation  fnun  juice  of  hard  chancre  showing 
spiroi -ha-tr  pallida.  (iiemsa's  stain,  x 2000.  (From  a  preparation 
liy  Dr.  Haswell  Wilson.) 

tin-  results  may  be  given.  In  the  primary  sore  and  in  the  related 
lymphatic  glands,  the  juice  of  which  can  be  conveniently 
obtained  by  means  of  a  hypodermic  syringe,  the  organism  has 
l>eeii  found  in  a  very  large  majority  of  cases.  It  has  been  also 
obtained  in  the  papular  and  roseolar  eruptions,  in  condylomata 
and  mucous  patches — in  fact,  one  may  say  generally,  in  all  the 
primary  and  secondary  lesions.  It  has  been  obtained  from 
the  splri'ii  during  life,  and  on  a  few  occasions,  e.g.  by  Schaudinn, 
a  No  from  the  blood  during  life  in  secondary  syphilis.  In  the 
congenital  form  of  the  disease  the  organism  may  be  present  in 
large  numbers  (Plate  II.,  Fig.  6),  as  was  first  shown  by  Buschke 
and  Fischer,  and  by  Levaditi.  In  the  pemphigoid  bullae,  in 


506  SYPHILIS 

the  blood,  in  the  internal  organs,  the  liver,  lungs,  spleen,  supra- 
renals,  and  even  in  the  heart  its  detection  may  be  comparatively 
easy,  owing  to  the  large  numbers  present  (Fig.  155).  It  has 
been  generally  supposed  that  tertiary  syphilitic  lesions  are  non- 
infective,  and  the  results  of  the  earlier  observations  on  the 
spirochsete  pallida  were  apparently  in  accordance  with  this 
view,  as  they  gave  negative  results.  More  prolonged  search 
has,  however,  shown  that  the  organism  may  occur  in  tertiary 
lesions  also.  It  has  been  found  to  be  present  in  the  peripheral 
parts  of  gummata,  especially  at  an  early  stage  of  their  forma- 


FIG.  155. — Section  of  spleen  from  a  case  of  congenital  syphilis,, 
showing  several  examples  of  spirochrete  pallida ;  Levaditi's  method, 
x  2000. 

tion ;  and  the  observations  of  Schmorl,  Benda,  J.  H.  Wright 
and  others  show  that  it  is  often  to  be  found  in  syphilitic 
aortitis,  sometimes  occurring  in  considerable  numbers  in  the 
thickened  patches.  That  the  spirochsete  may  persist  in  the 
body  for  a  very  long  time  after  infection,  has  been  abund- 
antly shown  by  different  observers ;  in  one  case,  for  example, 
its  presence  was  demonstrated  sixteen  years  after  the  primary 
lesion.  It  can  readily  be  demonstrated  in  sections  of  the  organs 
by  the  method  described  on  p.  112.  In  such  preparations  large 
numbers  of  spirochsetes,  chiefly  extra-vascular  in  position,  can 
be  seen,  and  many  may  occur  in  the  interior  of  the  more  highly 
specialised  cells,  for  example,  liver-cells ;  in  many  cases  examina- 


CULTIVATION  OF  THE  SPIROCH^ETE  PALLTDA  507 

tion  has  been  made  within  so  short  a^"  period  after  the  death  of 
the  child  as  to  practically  exclude  the  possibility  of  contamina- 
tion from  without.  It  also  abounds  sometimes  on  mucous 
surfaces,  e.</.  of  the  bladder  and  intestine  in  cases  of  congenital 
syphilis.  The  enormous  numbers  of  the  organism  which  may 
be  present  in  a  well  preserved  condition  in  macerated  foetuses 
render  it  probable  that  the  organism  may  multiply  in  the  dead 
tissues  under  anaerobic  conditions.  Although  various  organisms 
may  be  associated  with  it  in  the  lesions  of  the  skin  or  mucous 
membranes,  then-  is  agreement  amongst  observers  that  this 
organism  occurs  alone  in  syphilitic  lesions  where  the  entrance 
of  bacteria,  etc.,  from  outside  is  excluded.  The  high  per- 
centage of  cases  in  which  it  is  found  would,  in  view  of  the 
(lilliculty  in  detecting  it,  point  to 
its  invariable  presence,  and,  as  a  ^|J 

matter  of    fact,    Schaiidinn   in    his 

last  .series  of  cases,  numbering  over      jm  •* 

seventy,   found  it   in   all.     Shortly 
after  the  discovery  of  the  organism,     . 
Metclmikott'  was  able  to  detect  it  in  m  • 

the  lesions  produced  in  monkeys  by 
inoculation  with  material  derived 
from  syphilitic  sores,  and  his  obser- 
vations have  since  been  confirmed. 
Another  question  of  considerable 

importance  is,   as  to   whether  this     Fj(;  K((.     Bpiro6hlete  ren.ingens 
organism  has  been    found  in   other        In  film  preparation  from  a  ewe 
conditions.        Observations       show        of  balanilis.     xlOOO. 
that  in  various  conditions,  such  as 

ulcerated  carcinomata,  balanitis,  etc.,  spirochaetes  are  of  com- 
paratively common  occurrence.  There  is  no  doubt  whatever 
that  the  great  majority  of  these  are  readily  distinguishable  by 
their  appearance  from  the  spirochsete  pallida,  but  others  re- 
semble it  closely.  Hoti'mann,  however1,  who  has  seen  many  of 
these  spirocha-tes  from  other  sources,  considers  that  even  by 
their  microscopic  api>earance  they  are  capable  of  being  distin- 
-uMied,  though  with  considerable  difficulty.  It  must,  of  course, 
be  borne  in  mind  that  the  finding  of  an  organism  in  non-syphilitic 
lesions  with  the  same  microscopical  characters  does  not  show 
that  it  is  the  same  organism  as  the  spirochaete  pallida. 

Cultivation. — Although  numerous  attempts  have  been  made, 
it  has  not  yet  been  found  possible  to  obtain  pure  cultures  of  the 
-pirochsBte  pallida  outside  the  body.  Levaditi  and  Mclntosh 
inoculated  with  syphilitic  material  sacs  of  collodion  containing 


508  SYPHILIS 

human  serum,  heated  at  60°C.,  and  placed  them  in  the  peritoneal 
cavity  of  a  monkey  (macacus  cynomolgus).  After  an  interval  of 
about  three  weeks,  they  found  in  the  sacs  an  abundant  growth 
of  spirochsetes  morphologically  identical  with  spirochsete  pallicla, 
along  with  various  anaerobic  bacteria.  They  were  able  to 
continue  such  cultures  in  like  conditions,  but  were  unable  to 
obtain  any  pathogenic  effects  on  inoculating  animals  with  the 
material  from  the  sacs,  and  considered  that  the  organisms  had 
became  avirulent  owing  to  their  conditions  of  growth.  Recently 
Schereschewsky  claims  to  have  obtained  impure  cultures  of  the 
organism  in  test  tubes.  He  used  for  this  purpose  horse  serum 
inspissated  at  58°  C.,  and  then  allowed  to  undergo  autolysis  for 
three  days  at  37°  C.  Although  abundant  growth  of  spirochaetes 
was  obtained,  he  was  unable  to  infect  animals  by  means  of  them, 
or  to  obtain  any  serum  reactions  which  would  go  to  show 
that  the  organism  was  really  the  spirochsete  pallida. 

Transmission  of  the  Disease  to  Animals. — Although  various 
experiments  had  previously  been  from  time  to  time  made  by 
different  observers,  in  some  cases  with  reported  successful  result, 
it  is  to  the  papers  of  Metchnikoff  and  Roux  (1903-5)  that  we 
owe  most  of  our  knowledge.  These  observers  have  carried  on  a 
large  series  of  observations,  and  have  shown  that  the  disease  can 
be  transmitted  to  various  species  of  monkey.  Of  those  the 
anthropoid  apes  are  most  susceptible,  the  chimpanzee  being  the 
most  suitable  for  experimental  purposes.  Their  results  have 
been  confirmed  by  Lassar,  Neisser,  Kraus,  and  others.  The 
number  of  experiments  on  these  animals  is  now  very  great,  and 
the  general  result  is  that  the  disease  has  been  transmitted  by 
material  from  all  the  kinds  of  syphilitic  lesions  in  which  spiro- 
chaetes  have  been  demonstrated,  including  even  the  blood  in 
secondary  syphilis  and  tertiary  lesions.  Inoculation  is  usually 
made  by  scarification  on  the  eyebrows  or  genitals ;  the  sub- 
cutaneous and  other  methods  of  inoculation  give  negative 
results.  The  primary  lesion  is  in  the  form  of  an  indurated 
papule  or  of  papules,  in  every  respect  resembling  the  human 
lesion.  Along  with  this  there  is  a  marked  enlargement  and 
induration  of  the  corresponding  lymphatic  glands.  The  primary 
lesion  appears  on  an  average  about  thirty  days  after  inoculation, 
and  secondary  symptoms  develop  in  rather  more  than  half  of  the 
cases  after  a  further  period  of  rather  longer  duration.  These 
are  of  the  nature  of  squamous  papules  on  the  skin,  mucous 
patches  in  the  mouth,  and  sometimes  palmar  psoriasis.  As  a 
rule,  the  secondary  manifestations  are  of  a  somewhat  mild 
degree,  and  in  no  instance  up  to  the  present  has  any  tertiary 


TRANSMISSION  OF  THE  DISEASE  TO  ANIMALS    509 

lesion  been  observed.  By  re-inoculation  from  the  lesions,  the 
disease  may  be  transferred  to  other  animals.  The  disease  may 
also  be  produced  in  baboons  and  macaques  (macacyj  sinicus  is 
one  of  the  most  susceptible),  but  these  animals  are  less  susceptible, 
and  secondary  manifestations  do  not  appear.  The  severity  of  the 
affection  amongst  aj>es  would  in  fact  appear  to  be  in  proportion  to 
the  nearness  of  the  relationship  of  the  animal  to  the  human 
subject. 

AJ  <ho\vn  tirst  by  Hansell,  and  more  recently  by  Bertarelli,  the 
eye  of  the  rabbit  is  susceptible  to  inoculation  from  syphilitic 
lesions.  The  material  used  is  introduced  in  a  finely  divided 
state  either  into  the  tissue  of  the  cornea  or  into  the  anterior 
chamber,  and  syphilitic  keratitis  or  iritis,  or  both,  may  result, 
there  being  a  period  of  incubation  of  at  least  two  weeks. 
Levaditi  and  Yamanouchi  have  recently  studied  the  stages  in 
detail,  and  find  that  the  spirochaetes  remain  in  the  inoculated 
material  unchanged  for  a  time ;  then  organisation  occurs  and 
the  spirocha3tes  multiply,  and  later  still  there  is  a  more  rapid 
multiplication  and  invasion  by  them  of  the  tissues  of  the  eye. 
The  period  of  incubation  is  thus  not  due  to  the  organism  passing 
through  some  cycle  of  development,  but  simply  to  its  requiring 
certain  conditions  for  multiplying  which  are  not  supplied  for 
s« mil-  time. 

The  production  of  the  disease,  experimentally,  has  supplied 
us  with  some  further  facts  regarding  the  nature  of  the  virus. 
It  has  been  shown  repeatedly  that  the  passage  of  fluid  con- 
taining the  virus  through  a  Berkefeld  filter  deprives  it  completely 
of  its  infectivity.  In  other  words,  the  virus  does  not  belong  to 
the  ultra-microscopic  group  of  organisms.  The  virus  is  also 
readily  destroyed  by  heat,  a  temperature  of  51°  C.  being 
fatal.  With  regard  to  the  production  of  immunity,  very  little 
of  a  satisfactory  nature  has  so  far  been  established.  It  has  been 
found  that  the  virus  from  a  macaque  monkey  produces  a  less 
severe  disease  in  the  chimpanzee  than  the  virus  from  the  human 
subject,  inasmuch  as  secondary  lesions  do  not  follow;  the  virus 
would  thus  appear  to  have  undergone  a  certain  amount  of 
attenuation  in  the  tissues  of  that  monkey.  The  effects  of  inject- 
ing emulsions  of  tertiary  lesions  or  of  serum  from  syphilitic 
patients,  at  the  time  of  inoculation  with  the  virus,  appear  to  be 
nil  ;  so  also  the  employment  of  the  virus  rendered  inactive  by 
heating  has  apparently  no  influence  in  acting  as  a  vaccine. 
There  is  some  evidence  that  the  serum  from  a  patient  suffering 
from  the  disease  when  mixed  with  the  virus  before  inoculation 
modifies  the  disease  to  a  certain  extent,  but  further  evidence  on 


510  SYPHILIS 

this  point  is  necessary.  As  mentioned  above,  the  spirochsete 
pallida  has  been  found  in  the  lesions  in  monkeys,  Metchnikoff 
and  Roux  obtaining  positive  results  in  more  than  75  per  cent, 
of  the  cases,  and  it  is  to  be  noted  that  here  also  the  organism 
has  been  found  deep  in  the  substance  of  the  papules,  un- 
accompanied by  any  other  organisms.  It  is  also  to  be  noted 
that  the  blood  of  infected  apes  after  a  time  gives  the  Wasser- 
mann  reaction. 

Serum  Diagnosis — Wassermann  Reaction. — The  method  of 
applying  this  test  has  already  been  given  (p.  131);  we  have  now 
to  consider  the  results  of  its  application.  There  is  general 
agreement  amongst  workers  at  the  subject  that  the  test  affords 
by  far  the  most  reliable  means  of  diagnosis  of  the  disease ;  and 
on  comparing  the  results  obtained  it  will  not  be  an  overestimate 
to  say  that  a  positive  result  may  be  obtained  in  at  least  90  per 
cent,  of  cases  where  there  is  evidence  of  active  general  infection. 
The  reaction  generally  appears  first  on  the  fifteenth  to  thirtieth 
day  after  appearance  of  the  sore,  and  then  gradually  becomes 
more  marked ;  during  the  period  of  secondary  manifestations  it 
is  practically  always  present ;  in  the  tertiary  stage  with  active 
manifestations  a  positive  result  is  only  a  little  less  frequent.  As 
the  disease  becomes  inactive  or  is  cured  the  reaction  may  disappear, 
but  it  is  to  be  noted  that  disappearance  of  the  reaction  after  being 
present  does  not  necessarily  imply  cure  of  the  disease.  It  may 
only  have  become  latent,  and  on  its  becoming  once  more  active 
the  reaction  may  re-appear.  Energetic  treatment  with  mercury 
may  also  diminish  or  annul  the  reaction ;  in  fact,  its  presence 
and  intensity  would  appear  to  be  definitely  related  to  the  activity 
of  the  syphilitic  lesions.  A  positive  reaction  is  also  present  in 
the  large  majority  of  cases  of  general  paralysis  and  of  tabes,  and 
may  be  given  by  the  cerebro-spinal  fluid  as  well  as  by  the  blood 
serum  in  such  cases.  As  regards  other  diseases,  a  positive 
reaction  has  been  recorded  as  occurring  in  leprosy  (p.  304)  and 
sleeping-sickness  and  also  in  yaws,  but  apart  from  these  diseases 
it  is  practically  never  met  with.  At  present  little  can  be  said  in 
explanation  of  the  Wassermann  reaction.  It  seems  to  depend 
on  the  interaction  of  lipoidal  substances  in  the  extract  with 
proteins  in  the  serum,  which  are  apparently  contained  in  the 
globulin  fraction ;  but  we  know  nothing  as  to  why  this  peculiar 
modification  of  the  serum  should  be  present  in  syphilis.  It  is 
now  generally  considered  that  it  does  not  depend  on  the  presence 
of  an  anti-substance  (immune-body),  which  in  association  with 
the  antigen  (the  spirochaite)  fixes  complement. 


I-'KAMIUESIA  OR  YAWS  511 

FRAMBCESIA  OR  YAWS. 

l-'rambuesia  is  a  contagious  disease  of  the  tropics,  occurring  in 
the  west  coast  of  Africa,  Ceylon,  the  West  Indies,  and  other 
parts.  It  is  characterised  by  a  peculiar  cutaneous  eruption,  and 
it  is  markedly  contagious.  Its  resemblance  in  many  respects 
t«»  syphilis  has  been  noted,  and  the  relation  of  the  two  diseases 
1ms  been  the  subject  of  much  controversy.  It  is  accordingly  a 
matter  of  great  interest  that  an  organism  of  closely  similar 
characters  to  the  spirochuete  pallida  has  been  found  in  the  lesions 
of  f  ramboesia.  This  organism  was  discovered  by  Oastellani,  who 
gave  to  it  the  name  spirochatte  pertenuis  or  pallidula.  Morpho- 
logically, it  is  practically  identical  with  the  spirochaete  pallida ; 
when  ulceration  has  occurred  other  spirochaetes  of  less  regular 
form  may  be  present  as  contaminations.  In  the  skin  lesions 
it  has  been  shown  by  Levaditi's  method  to  be  present  in  con- 
siderable numbers,  especially  in  the  epidermis  and  also  amongst 
the  leucocytic  infiltration,  which  comprises  more  polymorpho- 
nuclear  leucocytes  than  is  seen  in  the  case  of  syphilis.  Castellani 
showed  that  the  disease  could  be  transferred  to  monkeys  (semno- 
jiltkccus  and  macacus  being  used  for  this  purpose),  and  that  the 
organism  could  be  demonstrated  in  the  unbroken  skin  lesions. 
The  lesions  are  as  a  rule  confined  to  the  site  of  inoculation,  but 
the  infection  is  general,  as  is  shown  by  the  presence  of  spirochaetes 
in  the  lymphatic  glands  and  the  spleen.  These  results  with 
regard  to  the  presence  of  spirochaete  pallidula  in  the  lesions  and 
the  inoculation  of  apes  have  been  confirmed  by  other  workers, 
and  the  etiological  relationship  of  the  organism  to  the  disease 
may  now  be  regarded  as  practically  established.  The  immunity 
reactions  in  monkeys  infected  with  syphilis  and  frambcesia,  as 
experimentally  studied  by  Castellani  and  by  Neisser,  Baermann, 
and  Halberstadter,  go  to  show  that  the  two  diseases  are  distinct. 
On  the  other  hand,  Levaditi  and  Nattan-Larrier  found  that, 
although  monkeys  infected  with  syphilis  were  refractory  to 
framboesia  (Fr.  pian\  monkeys  infected  with  frambcesia  were  sus- 
cvptiblr  to  syphilis:  they  therefore  concluded  that  frambcesia 
is  a  modified  or  mild  form  of  syphilis.  The  exact  relationship 
of  the  two  diseases  cannot  be  yet  accurately  defined,  but  they 
are  undoubtedly  closely  related,  and  probably  have  a  common 
parentage. 


CHAPTER   XXL 

IMMUNITY. 

Introductory. — By  immunity  is  meant  non-susceptibility  to  a 
given  disease  or  to  a  given  organism,  either  under  natural 
conditions  or  under  conditions  experimentally  produced.  The 
term  is  also  used  in  relation  to  the  toxins  of  an  organism. 
Immunity  may  be  possessed  by  an  animal  naturally,  and  is  then 
usually  called  natural  immunity,  or  it  may  be  acquired  by  an 
animal  either  by  its  passing  through  an  attack  of  the  disease,  or 
by  means  of  artificial  inoculation.  It  is  to  be  noted  that  man  and 
the  lower  animals  may  be  exempt  from  certain  diseases  under 
natural  conditions,  and  yet  the  causal  organisms  of  these  diseases 
may  produce  pathogenic  effects  when  injected  in  sufficient 
quantity.  Immunity  is,  in  fact,  of  very  varying  degrees,  and 
accordingly  the  use  of  the  term  has  a  correspondingly  relative 
significance.  This  is  not  only  true  of  infection  by  bacteria,  but 
of  toxins  also  :— when  the  resistance  of  an  animal  to  these  is  of 
high  degree,  the  resistance  may  in  certain  cases  be  overcome  by 
a  very  large  dose  of  the  toxic  agent.  On  the  other  hand,  in 
cases  where  the  natural  powers  of  resistance  are  very  high, 
these  can  be  still  further  exalted  by  artificial  means,  that  is, 
the  natural  immunity  may  be  artificially  intensified. 

Acquired  Immunity  in  the  Human  Subject. — The  following 
facts  are  supplied  by  a  study  of  the  natural  diseases  which  affect 
the  human  subject.  First,  in  the  case  of  certain  diseases,  one 
attack  protects  against  another  for  many  years,  sometimes 
practically  for  a  lifetime,  e.g.  smallpox,  typhoid,  scarlet  fever, 
etc.  Secondly,  in  the  case  of  other  diseases,  e.g.  erysipelas, 
diphtheria,  influenza,  and  pneumonia,  a  patient  may  suffer  from 
several  attacks.  In  the  case  of  the  diseases  of  the  second  group, 
however,  experimental  research  has  shown  that  in  many  of 
them  a  certain  degree  of  immunity  does  follow;  and,  though 
we  cannot  definitely  state  it  as  a  universal  law,  it  must  be 
considered  highly  probable  that  the  passing  through  an  attack 

612 


ARTIFICIAL  IMMUNITY  513 

of  an  acute  disease  produced  by  an  organism,  confers  immunity 
for  a  longer  or  shorter  period.  The  immunity  is  not,  however, 
to  be  regarded  as  the  result  of  the  disease  per  se,  but  of  the 
bacterial  products  introduced  into  the  system;  as  will  be  shown 
ln-l«i\v,  by  suitable  gradation  of  the  doses  of  such  products,  or 
by  the  use  of  weakened  toxins,  a  high  degree  of  immunity  may 
be  attained  without  the  occurrence  of  any  symptoms  whatever. 

The  facts  known  regarding  vaccination  and  smallpox  exemplify 
another  principle.  We  may  take  it  as  practically  proved  that 
vaccinia  is  variola  or  smallpox  in  the  cow,  and  that  when 
vaccination  is  performed,  the  patient  is  inoculated  with  a 
modified  variola  (vide  Smallpox,  in  Appendix).  Vaccination 
produces  certain  pathogenic  effects  which  are  of  trifling  degree 
as  compared  with  those  of  smallpox,  and  we  find  that  the  degree 
of  protection  is  less  complete  and  lasts  a  shorter  time  than  that 
produced  by  the  natural  disease.  Again,  inoculation  with  lymph 
from  a  smallpox  pustule  produces  a  form  of  smallpox  less 
severe  than  the  natural  disease  but  a  much  more  severe  con- 
dition than  that  produced  by  vaccination,  and  it  is  found  that 
the  decree  of  protection  .or  immunity  resulting  occupies  an 
intermediate  position. 

I iniinniity  and  Recovery  from  Disease. — Recovery  from  an 
acute  infective  disease  shows  that  in  natural  conditions  the  virus 
may  be  exhausted  after  a  time,  the  period  of  time  varying  in 
different  diseases.  How  this  is  accomplished  we  do  not  yet 
fully  know,  but  it  has  been  found  in  the  case  of  diphtheria, 
typhoid,  cholera,  pneumonia,  etc.,  that  in  the  course  of  the 
disease  certain  substances  (called  by  German  writers  Antikorper) 
appear  in  the  blood,  which  are  antagonistic  either  to  the  toxin 
or  to  the  vital  activity  of  the  organism.  In  such  cases  a  process 
of  immunisation  would  appear  to  be  going  on  during  the  pro- 
of the  disease,  and  when  this  immunisation  has  reached  a 
certain  height,  the  disease  naturally  comes  to  an  end.  It  cannot, 
however,  be  said  as  yet  that  such  antagonistic  substances  are 
developed  in  all  cases ;  though  the  results  already  obtained  make 
this  probable. 

ARTIFICIAL  IMMUNITY. 

Varieties. — According  to  the  means  by  which  it  is  produced, 
immunity  may  be  said  to  be  of  two  kinds,  to  which  the  terms 
'I'-fitte  and  passive  are  generally  applied,  or  we  may  speak  of 
immunity  directly,  or  indirectly,  produced.  We  shall  first  give 
an  account  of  the  established  facts,  and  afterwards  discuss  some 

33 


514  IMMUNITY 

of  the  theories  which  have  been  brought  forward  in  explanation 
of  these  facts. 

Active  immunity  is  obtained  by  (a)  injections  of  the  organisms 
either  in  an  attenuated  condition  or  in  sub-lethal  doses,  or  (b) 
by  sub-lethal  doses  of  their  products,  i.e.  of  their  "toxins,"  the 
word  being  used  in  the  widest  sense.  By  repeated  injections 
at  suitable  intervals  the  dose  of  organisms  or  of  the  products 
can  be  gradually  increased ;  or,  what  practically  amounts  to  the 
same,  an  organism  of  greater  virulence  or  a  toxin  of  greater 
strength  may  be  used.  A  degree  of  resistance  or  immunity 
can  thus  be  developed,  and  this  in  course  of  time  may  reach 
a  very  high  level.  Such  methods  constitute  the  means  of 
preventive  inoculation  or  vaccination.  Immunity  of  this  kind 
is  comparatively  slowly  produced  and  lasts  a  considerable  time, 
the  duration  varying  in  different  cases.  The  principles  of 
vaccination  have  within  recent  years  been  extended  by  Wright 
to  the  treatment  of  disease. 

Passive  immunity  depends  upon  the  fact  that  if  an  animal 
be  immunised  to  a  very  high  degree  by  the  previous  method,  its 
serum  may  have  distinctly  antagonistic  or  neutralising  effects 
when  injected  into  another  animal  along  with  the  organisms,  or 
with  their  products,  as  the  case  may  be.  Such  a  serum, 
generally  known  as  an  anti-serum,  may  exert  its  effects  if  intro- 
duced into  an  animal  at  the  same  time  as  infection  occurs  or 
even  a  short  time  afterwards ;  it  can,  therefore,  be  employed 
as  a  curative  agent.  The  serum  is  also  preventive,  i.e.  protects 
an  animal  from  subsequent  infection,  but  the  immunity  thus 
conferred  lasts  a  comparatively  short  time.  These  facts  form 
the  basis  of  serum  therapeutics.  When  such  a  serum  has  the 
power  of  neutralising  a  toxin  it  is  called  antitoxic  ;  when,  with 
little  or  no  antitoxic  power,  it  protects  against  the  living 
bacterium  in  a  virulent  condition,  it  is  called  antimicrobic  or 
antibacterial  (vide  infra). 

In  the  accompanying  table  a  sketch  of  the  chief  methods  by 
which  an  immunity  may  be  artificially  produced  is  given.  It 
has  been  arranged  merely  for  purposes  of  convenience  and  to 
aid  subsequent  description ;  the  principles  underlying  all  the 
methods  are  the  same. 


ARTIFICIAL  IMMUNITY. 

A.  Active  Immunity — i.e.  produced  in  an  animal  by  an  in- 
jection, or  by  a  series  of  injections,  of  non-lethal  doses  of 
an  organism  or  its  toxins. 


ACTIVE  IMMUNITY  515 

1.  By  injection  of  the  Hiring  organisms. 

(a)  Attenuated  in  various  ways.     Examples  : — 

(1)  By  growing  in  the  presence  of  oxygen,  or  in  a 

current  of  air. 

(2)  By   passing   through   the   tissues   of   one   species 

of    animal    (becomes   attenuated    for    another 
species). 

(3)  By  growing  at  abnormal  temperatures,  etc. 

(4)  By  growing  in  the  presence  of  weak  antiseptics,  or 

by  injecting  the  latter  along  with  the  organism, 
etc. 

(b)  In  a  virulent  condition,  in  non-lethal  doses. 

2.  By  injection  of  the  dead  organisms. 

3.  By  injection  of  jiltered  bacterial  cultures,  i.e.  toxins  ;  or  of 

substances  derived  from  such  filtrates. 
These  methods  may  also  be  combined  in  various  ways. 

B.  Passive  Immunity,  i.e.  produced  in  one  animal  by  injection 
of  the  serum  of  another  animal  highly  immunised  by  the 
methods  of  A. 

1.  By  antitoxic  serum,  i.e.  the  serum  of  an  animal  highly 

immunised  against  a  particular  toxin. 
'2.    It  a  antibacterial  serum,  i.e.  the  serum  of  an  animal  highly 

immunised  against  a  particular  bacterium  in  the  living 

and  virulent  condition. 

A.  Active  Immunity. 

1.  By  Living  Cultures. — (a)  Attenuated. — In  the  earlier 
work  on  immunity  in  the  case  of  anthrax,  chicken  cholera,  swine 
plague,  etc.,  the  investigators  had  to  deal  with  organisms  of 
high  virulence,  which  had  accordingly  to  be  reduced  before  the 
organisms  could  be  injected  in  the  living  state.  It  is  now  found 
most  convenient  a£  a  rule  to  start  the  process  of  active  immunisa- 
tion with  the  injection  of  dead  cultures.  The  principle  is  the 
same  as  that  of  vaccination,  and  both  attenuated  cultures  and 
also  the  dead  cultures  used  for  injection  are  often  spoken  of  as 
rtirrfnes.  The  virulence  of  an  organism  may  be  diminished  in 
various  ways,  of  which  the  following  examples  may  be  given  : — 

(1)  In  the  first  place,  practically  every  organism,  when  culti- 
vated for  some  time  outside  the  body,  loses  its  virulence,  and  in 
the  case  of  some  this  is  very  marked  indeed,  e.g.  the  pneumo- 
coccus.  Pasteur  found  in  the  case  of  chicken  cholera,  that 


516  IMMUNITY 

when  cultures  were  kept  for  a  time  in  ordinary  conditions,  they 
gradually  lost  their  virulence,  and  that  when  sub-cultures  were 
made  the  diminished  virulence  persisted.  Such  attenuated 
cultures  could  be  used  for  protective  inoculation.  He  considered 
the  loss  of  virulence  to  be  due  to  the  action  of  the  oxygen  of 
the  air,  as  he  found  that  in  tubes  sealed  in  the  absence  of  oxygen 
the  virulence  was  not  lost.  Haffkine  attenuated  cultures  of  the 
cholera  spirillum  by  growing  them  in  a  current  of  air  (p.  459). 

('2)  The  virulence  of  an  organism  for  a  particular  animal  may 
be  lessened  by  passing  the  organism  through  the  body  of  another 
animal.  Duguid  and  Burdon  Sanderson  found  that  the  virulence 
of  the  anthrax  bacillus  for  bovine  animals  was  lessened  by  its 
being  passed  through  guinea-pigs,  the  disease  produced  in  the 
ox  by  inoculation  from  the  guinea-pig  being  a  non-fatal  one. 
This  discovery  was  confirmed  by  Greenfield,  who  showed  that 
the  bacilli  cultivated  from  guinea-pigs  preserved  their  property 
in  cultures,  and  could  therefore  be  used  for  protective  inoculation 
of  cattle.  A  similar  principle  was  applied  in  the  case  of  swine 
plague  by  Pasteur,  who  found  that  if  the  organism  producing 
this  disease  was  inoculated  from  rabbit  to  rabbit,  its  virulence 
was  increased  for  rabbits  but  was  diminished  for  pigs.  The 
method  of  vaccination  against  small-pox  depends  upon  the  same 
principle.  There  is  also  evidence  to  show  that  the  virulence  of 
the  tubercle  bacillus  becomes  modified  according  to  its  host, 
being  often  diminished  for  other  animals. 

(3)  Many  organisms  become    diminished    in  virulence  when 
grown    at    an   abnormally   high   temperature.     The  method  of 
Pasteur,    already   described  (p.   346),  for  producing    immunity 
in  sheep  against    anthrax  bacilli,  depends  upon  this  fact.     A 
virulent  organism  may  also  be  attenuated  by  being  exposed  to 
an  elevated  temperature  which  is  insufficient  to  kill  it,  as  was 
found  by  Toussaint  in  the  case  of  anthrax. 

(4)  Still   another    method  may  be    mentioned,    namely,    the 
attenuation  of  the  virulence  by  growing   the    organism  in  the 
presence    of   weak  antiseptics.       Chamberland   and   Roux,    for 
example,    succeeded    in    attenuating   the    anthrax   bacillus    by 
growing  it  in  a  medium  containing  carbolic  acid  in  the  propor- 
tion of  1  :  600. 

These  examples  will  serve  to  show  the  principles  underlying 
attenuation  of  the  virulence  of  an  organism.  There  are,  how- 
ever, still  other  methods,  most  of  which  consist  in  growing  the 
organism  in  conditions  somewhat  unfavourable  to  its  growth,  e.g. 
under  compressed  air,  etc. 

(6)    Immunity   by   living    Virulent    Cultures   in    Non-lethal 


BY  LIVING  CULTURES  517 

Doses. — Immunity  may  also  be  produced  by  employing  virulent 
cultures  in  small,  that  is  non-lethal,  doses.  In  subsequent 
inoculations  the  doses  may  be  increased  in  amount.  For 
example,  immunity  may  thus  be  obtained  in  rabbits  against  the 
bacillus  pyocyaneus.  Such  a  method,  however,  lias  had  only  a 
limited  application,  as  it  has  been  found  more  convenient  to 
commence  the  process  by  dead  or  attenuated  cultures,  and  then 
to  continue  with  virulent  cultures. 

Exaltation  of  the  Virulence. — The  converse  process  to  attenua- 
tion, i.e.  the  exaltation  of  the  virulence,  is  obtained  chiefly  by 
the  method  of  cultivating  the  organism  from  animal  to  animal — 
the  method  of  passage  discovered  by  Pasteur  (first,  we  believe, 
in  the  case  of  an  organism  obtained  from  the  saliva  in  hydro- 
phobia, though  having  no  causal  relationship  to  that  disease). 
This  is  most  conveniently  done  by  intraperitoueal  injections,  as 
there  is  less  risk  of  contamination.  The  organisms  in  the 
peritoneal  fluid  may  be  used  for  the  subsequent  injection,  or  a 
culture  may  l>e  made  between  each  inoculation.  The  virulence 
of  a  great  number  of  organisms  can  be  increased  in  this  way, 
the  animals  most  frequently  used  being  rabbits  and  guinea-pigs. 
This  method  can  be  applied  to  the  organisms  of  typhoid,  cholera, 
pneumonia,  to  streptococci  and  staphylococci,  and  in  fact  to 
those  organisms  generally  which  invade  tissues. 

The  virulence  of  an  organism,  especially  when  in  a  relatively 
attenuated  condition,  can  also  be  raised  by  injecting  along  with 
it  a  quantity  of  a  culture  of  another  organism  either  in  the  living 
or  dead  condition.  A  few  examples  may  be  mentioned.  An 
attenuated  diphtheria  culture  may  have  its  virulence  raised  by 
being  injected  into  an  animal  along  with  the  streptococcus 
pyogenes ;  an  attenuated  culture  of  the  bacillus  of  malignant 
oedema  by  being  injected  with  the  bacillus  prodigiosus;  an 
attenuated  streptococcus  by  being  injected  with  the  bacillus  coli, 
etc.  A  culture  of  the  typhoid  bacillus  may  be  increased  in 
virulence,  as  already  stated,  by  being  injected  along  with  a  dead 
culture  of  the  bacillus  coli.  In  such  cases  the  accompanying 
injection  enables  the  attenuated  organism  to  gain  a  foothold  in 
the  tissues,  and  it  may  be  stated  as  a  general  rule  that  the 
virulence  of  an  organism  for  a  particular  animal  is  raised  by  its 
growing  in  the  tissues  of  that  animal. 

Combination  of  Methods. — The  above  methods  may  be  com- 
bined in  various  ways.  By  repeated  injections  of  cultures  at 
first  in  the  dead  condition,  then  living  and  attenuated  and 
afterwards  more  virulent,  and  by  increasing  the  doses,  a  high 
degree  of  immunity  may  be  obtained. 


518  IMMUNITY 

2.  Immunity  by  Dead  Cultures  of  Bacteria. — In  some  cases 
a  high  degree  of  immunity  against  infection  by  a  given  microbe 
may  be  developed  by  repeated  and  gradually  increasing  doses 
of  the  dead  cultures,  the   cultures   being   killed  sometimes  by 
heat,  sometimes  by  exposure  to  the  vapour  of  chloroform.     In 
this  method  the  so-called  endotoxins  will  be  injected  along  with 
the  other  substances  in  the  bacterial  protoplasm,  but  the  result- 
ing immunity  is  chiefly  directed  against  the  vital  activity  of  the 
organisms — is  antibacterial  rather  than    antitoxic  (vide  infra). 
The  cultures  when  dead  produce,  of  course,  less  effect  than  when 
living,  and  this  method  may  be  conveniently  used  in  the  initial 
stages  of  active  immunisation, — to  be  afterwards    followed  by 
injections  of   the  living   cultures.     The    method  is   extensively 
used  for  experimental  purposes,  and  is  that  adopted  in  anti-plague 
and  anti-typhoid  inoculations,  and  in  the  treatment  of  infections 
by  means  of  vaccines. 

3.  Immunity    by    the    Separated    Bacterial    Products   or 
Toxins. — The  organisms  in  a  virulent  condition  are  grown  in 
a  fluid  medium  for  a  certain  time,  and  the  fluid  is  then  filtered 
through  a  Chamberland  or  other  porcelain  filter.     The  filtrate 
contains  the  toxins,  and  it  may  be  used  unaltered,  or  may  be 
reduced  in  bulk  by  evaporation,  or  may  be  evaporated  to  dryness. 
The  process  of  immunisation  by  the  toxin  is  started  by  small 
non-lethal  doses  of  the  strong  toxin,  or  by  larger  doses  of  toxin 
the  power  of  which  has  been  weakened  by  various  methods  (vide 
infra).     Afterwards    the  doses  are  gradually    increased.     This 
method  was  carried  out  with  a  great  degree  of  success  in  the 
case  of  diphtheria,  tetanus,  malignant  oedema,  etc.     It  appears 
capable  of  general  application  in  the  case  of  organisms  where  it 
is  possible  to  get  an  active  toxin  from  the  filtered  cultures.     It 
has  also  been  applied  in  the  case  of  snake  venoms  by  Calmette 
and   by   Fraser,    and    a   high    degree    of    immunity    has   been 
produced. 

The  following  may  be  mentioned  as  some  of  the  most 
important  examples  of  the  practical  application  of  the  principles 
of  active  immunity,  i.e.  of  protective  inoculation  : — (1)  Inocula- 
tion of  sheep  and  oxen  against  anthrax  (Pasteur)  (p.  346) ;  (2) 
Jenneriau  vaccination  against  smallpox  (p.  565) ;  (3)  Anti- 
cholera  inoculation  (Haffkine)  (p.  459)  ;  (4)  Anti-plague 
inoculation  (Haffkine)  (p.  486) ;  (5)  Anti-typhoid  inoculation 
(Wright  and  Semple)  (p.  375)  ;  (6)  Pasteur's  method  of  inocula- 
tion against  hydrophobia,  which  involves  essentially  the  same 
principles  (p.  579). 

Vaccines  as  a  Method  of   Treatment. — Up  till    recently  the 


BY  BACTERIAL  PRODUCTS  OR  TOXINS        519 

principles  of  active  immunity  had  not  been  directly  applied  in 
the  treatment  of  an  existing  disease  except  in  the  case  of  tuber- 
culosis.    The   work   of    Wright,    however,    shows    that   active 
immunisation  in  such  circumstances  is  not  only  possible,  but  is 
also  probably  capable  of  wide  application.     From  his  study  of 
the  part  played  by  phagocytosis   in   the   successful  combat  of 
bacteria  by  the  body,  he  was  led  to  advocate  the  treatment 
of  bacterial  infections  by  carrying  on  an  active  immunisation 
against  the  causal  agents  by  the  injection  of  dead  cultures  of 
the  latter.     The  justification  for  such  a  procedure   lies  in  his 
contention  that  in  many  cases  infections  are  to  be  looked  on  as 
practically  localised,  e.g.  the  cases  of  an  acne  pustule,  or  a  boil. 
The  reason  for  the  local  growth  of  bacteria  in  the  part  of  the 
body  affected  is  that  there  is  for  unknown  causes  a  deficiency 
of  the  opsonic  power  (vide  p.  291)  of  the  body  fluids,  which  is 
essential  for  the  phagocytosis  of   the  invading  bacteria.     Still 
more    marked    in  such  cases  is  the   deficiency  in    the   opsonic 
qualities   of  the   fluids   in   the   actual  site  of   infection.     Any 
procedure  which  will  raise  the  opsonic  power  of  the  body  fluids 
M  ;i  whole,  and  therefore  of  the  fluids  in  the  focus  of  infection, 
will  aid  the  destruction  of  the  bacteria  by  sensitising  them  to 
phagocytic  action.     Such  a  procedure   is   found   in  the  active 
immunisation  which   results  from   the   injection  of   a    vaccine 
consisting   of   a   dead    culture    of    the   causal    bacteria.      The 
application   of   a  vaccine  of   this   kind   can  be  controlled   by 
observation  of  the  opsonic  index  of  the  patient's  serum  during 
the  treatment.     When  a  local  infection  is  present  the  general 
opsonic  index  is  usually  found   to   be  below  unity.     If   dead 
bacteria  be  injected  into  the  individual  there  may  occur  during 
the  following  few  days  a  further  fall  in  the  opsonic  index, — 
what  Wright  calls  the  occurrence  of  a  negative  phase.  •  In  a  case 
where  the  treatment  is  successful,  this  negative  phase  is  succeeded 
by  a  rise  in  the  opsonic  index  above  its  original  level, — occurrence 
of  positive  phase, — and  with  this  reaction  there  is  an  improve- 
ment in   the  local  condition.     Usually  in  such  cases  repeated 
injections  are  required  to  effect  a  cure,  and  the  important  point, 
according  to  Wright,  is   to   avoid  giving  an  injection  when  a 
negative  phase  is  in  progress.     If  this  point  is  not  attended  to 
an    aggravation    of    symptoms    may    occur     (vide    p.     291). 
With  regard  to  the  details  of  the  preparation  of  the  vaccines  see 
p.  133.     The  number  of  bacteria  employed  for  a  vaccination 
varies  from  5,000,000  to  500,000,000  or  more.     Such  vaccines 
have  been  used   extensively  in    the    treatment   of   acne,    boils, 
sycosis,  tuberculosis,  infections  of  the  genito-urinary  tract  by  the 


520  IMMUNITY 

b.  coli,  infections  of  joints  by  the  gonococcus,  and  in  many  cases 
considerable  success  has  followed  the  treatment. 

Active  Immunity  by  Feeding. — Ehrlich  found  that  mice 
could  be  gradually  immunised  against  ricin  and  abrin  by  feeding 
them  with  increasing  quantities  of  these  substances  (vide  p.  199). 
In  the  course  of  some  weeks'  treatment  in  this  way  the  resulting 
immunity  was  of  so  high  a  degree  that  the  animals  could  tolerate 
on  subcutaneous  inoculation  400  times  the  dose  originally  fatal. 
Fraser  also  found  in  the  case  of  snake  venom  that  rabbits  could, 
by  feeding  with  the  poison,  be  immunised  against  several  times 
the  lethal  dose  of  venom  injected  into  the  tissues. 

By  feeding  animals  with  dead  cultures  of  bacteria  or  with 
their  separated  toxins,  a  degree  of  immunity  may  in  some  cases 
be  gradually  developed.  But  this  method  is  so  much  less  certain 
in  results,  and  so  much  more  tedious  than  the  others,  that  it  has 
obtained  no  practical  applications. 

Active  immunity  of  high  degree  developed  by  the  methods 
described  may  be  regarded  as  specific,  that  is,  is  exerted  only 
towards  the  organism  or  toxin  by  means  of  which  it  has  been 
produced.  A  certain  degree  of  immunity,  or  rather  of  increased 
general  resistance  of  parts  of  the  body  (for  example  the  peri- 
toneum), can,  however,  be  produced  by  the  injection  of  various 
substances — bouillon,  blood  serum,  solution  of  nuclein,  etc. 
(Issaeff).  Also  increased  resistance  to  one  organism  can  be  thus 
produced  by  injections  of  another  organism.  Immunity  of  this 
kind,  however,  never  reaches  a  high  degree. 

B.  Passive  Immunity. 

Action  of  the  Serum  of  Highly  Immunised  Animals. — 1. 
The  serum  of  an  animal  A,  treated  by  repeated  and  gradually 
increased  doses  of  a  toxin  of  a  particular  microbe,  may  protect 
an  animal  B  against  a  certain  amount  of  the  same  toxin  when 
injected  along  with  the  latter,  or  a  short  time  before  it.  As 
might  be  expected,  it  has  less  effect  when  injected  some  time 
afterwards,  but  even  then  within  certain  limits  it  has  a  degree 
of  curative  or  palliative  power.  Seeing  that  the  serum  of  animal 
A  appears  to  neutralise  the  toxin,  the  term  antitoxic  has  been 
applied  to  it. 

2.  The  serum  of  an  animal  A,  highly  immunised  against  a 
bacterium  by  repeated  and  gradually  increasing  doses  of  the 
organism,  may  protect  an  animal  B  against  an  infection  by 
the  living  organism  when  injected  under  conditions  similar  to  the 
above.  This  serum  is  therefore  antimicrobic,  or  antibacterial, 


PASSIVE  IMMUNITY  521 

i.e.  preventive  against  invasion  by  a  particular  organism.  (In 
addition  to  the  preventive  or  protective  action  in  vivo,  such  a 
serum  may  exert  certain  recognisable  effects  on  the  corresponding 
organism  in  vitro.  Thus  (a)  it  may  lead  to  the  death  or  solution 
of  the  organism — bactericidal  or  lysogenic  action ;  when  no  such 
effect  follows,  the  presence  of  an  immune-body  (p.  128)  may  be 
shown  by  the  deviation  of  complement  method ;  (b)  it  may  pro- 
duce an  increased  susceptibility  to  ingestion  by  phagocytes — 
opsonic  action ;  (c)  it  may  lead  to  the  clumping  of  the  organism 
— agglutinative  action,  or  to  precipitation  with  an  extract  of  a 
culture  of  the  corresponding  bacterium.) 

Anti-substances  and  their  Specificity. — The  fundamental  fact 
in  passive  immunity,  namely,  that  immunity  can  be  transferred  to 
another  animal,  shows  that  the  serum  in  question  differs  from 
the  serum  of  a  normal  animal  in  containing  antagonistic  sub- 
stances to  the  toxin  or  bacterium  as  the  case  may  be, — these 
being  generally  spoken  of  as  anti-substances.  The  development 
of  these  bodies,  first  observed  in  the  case  of  the  injection  *  of 
toxins,  is  found  to  occur  when  a  great  many  different  substances 
are  introduced  into  the  tissues  of  the  living  body.  We  can,  in 
fact,  divide  organic  molecules  into  two  classes — those  which  give 
rise  to  the  production  of  anti-substances,  and  are  thus  known  as 
antigens,  and  those  which  have  not  this  property.  Amongst  the 
former  are  various  toxins,  ferments,  molecules  of  tissue  cells, 
bacteria,  red  corpuscles,  etc.  They  are  all  probably  of  proteid 
nature,  though  their  true  constitution  is  not  known,  and  none  of 
them  have  been  obtained  in  a  pure  condition.  Amongst  the 
latter  may  be  placed  the  various  poisons  of  known  constitution, 
glncosides,  alkaloids,  etc.  We  may  also  state  at  present  that  the 
anti-substance  forms  a  chemical  or  physical  union  with  the 
particular  antigen  which  has  led  to  its  development,  and  we 
shall  discuss  the  evidence  for  this  later.  Furthermore,  the  anti- 
substance  has  apparently  a  specific  combining  group  which  fits,  as 
it  were,  a  group  in  the  corresponding  antigen,  the  two  groups 
having  been  compared  to  a  lock  and  key.  It  is,  however,  to  be 
noted  that  this  specificity  is  a  chemical  one  rather  than  a 
biological  one.  An  anti-serum,  for  example,  developed  by  the 
injection  of  bacterium  A  may  also  have  some  effect  on  bacterium 
B,  and  thus  appear  not  to  be  specific.  We  have,  however, 
evidence  to  show  that  the  antigens  in  bacterium  A  are  not  all 
identical,  and  that  some  of  them  may  be  present  though  in  smaller 
proportion  in  bacterium  B ;  thus  the  theory  of  combining  sjjeci- 
t'n-ity  is  not  invalidated.  The  number  of  different  anti-substances, 
as  judged  by  their  combining  properties,  would  appear  to  be  almost 


522  IMMUNITY 

unlimited,  a  fact  which  throws  new  light  on  the  complexity  of 
the  structure  of  living  matter.  When  anti-substances  are  studied 
as  regards  their  action  on  the  substances  with  which  they  com- 
bine, they  may  be  conveniently  arranged  in  three  classes 
corresponding  to  Ehrlich's  three  classes  of  receptors  (vide  p.  549). 
In  the  first  place,  the  anti-substance  may  simply  combine  with 
the  substance  without,  so  far  as  we  know,  producing  any  change 
in  it,  and  to  this  group  the  anti-toxins  and  anti-ferments  belong. 
In  the  second  place,  the  anti-substance,  in  addition  to  combining, 
may  produce  some  recognisable  physical  alteration.  In  other 
words,  it  possesses  an  active  or  zymotoxic  group  as  well  as 
a  combining  group.  The  agglutinins  may  be  mentioned  as 
examples  of  this  group.  In  the  third  place,  the  anti-substance 
after  combination  may  lead  to  the  combination  of  another  body 
normally  present  in  serum  called  complement  or  alexine,  and 
this  latter,  which  has  a  constitution  very  similar  to  that  of  a  toxin, 
may  lead  to  physical  change,  for  example,  death  or  solution  of  a 
cell.  Anti-substances  of  this  class  are  known  as  immune-bodies 
or  amboceptors  (Ehrlich)  or  as  sensitising  substances — substances 
sensibilisatrices  of  French  writers.  Their  essential  feature  is  that 
they  lead  to  the  combination  or  fixation  of  complement,  which 
may  or  not  produce  some  recognisable  change  such  as  bacterio- 
lysis, etc.  If  no  such  effect  follows,  however,  the  union  of  com- 
plement may  be  demonstrated  by  the  indirect  or  deviation 
method  (p.  130). 

After  this  preliminary  statement  in  explanation,  we  shall  con- 
sider the  actual  properties  of  the  two  classes  of  serum,  and  later 
we  shall  resume  the  theoretical  consideration. 

Antitoxic  Serum. — In  a  previous  chapter  (p.  188)  a  distinction 
has  been  drawn  between  extra-  and  intra-cellular  toxins,  and 
with  regard  to  these  the  general  statement  may  be  made  that 
while  antitoxins  are,  as  a  rule,  comparatively  easily  obtained  in 
the  case  of  the  former,  the  matter  is  quite  otherwise  in  the  case 
of  the  latter.  In  fact  some  writers  have  gone  so  far  as  to  say 
that  antitoxins  to  endotoxins  cannot  be  obtained.  Such  an 
extreme  view  is  in  our  opinion  unjustifiable  in  the  light  of  the 
recent  work  on  antitoxins  to  the  typhoid,  cholera,  and  dysentery 
endotoxins  (pp.  366,  456,  388).  Nevertheless  we  have  the  im- 
portant fact  that  in  many  cases  by  the  injection  of  dead  cultures 
an  active  anti-bacterial  serum  can  be  obtained  which  has  no 
neutralising  action  on  the  endotoxins,  and  we  must  conclude 
either  that  a  large  proportion  of  the  endotoxin  does  not  lead  to  the 
production  of  antitoxin  or  does  so  only  with  great  slowness,  the 
latter  alternative  being  on  general  grounds  rather  improbable. 


ANTITOXIC  SERUM  523 

The  best  examples  of  antitoxic  sera  are  those  of  diphtheria  and 
tetanus,  though  similar  principles  and  methods  are  involved  in 
the  case  of  the  anti-sera  to  ricin  and  abrin,  and  to  snake  poison. 
\\V  shall  here  speak  of  diphtheria  and  tetanus.  The  steps  in  the 
process  of  preparation  may  be  said  to  be  the  following  :  First, 
the  preparation  of  a  powerful  toxin ;  second,  the  estimation  of 
the  power  of  the  toxin ;  third,  the  development  of  antitoxin  in 
the  blood  of  a  suitable  animal  by  gradually  increasing  doses  of 
the  toxin ;  fourth,  the  estimation  from  time  to  time  of  the 
antitoxic  power  of  the  serum  of  the  animal  thus  treated. 

1.  Preparation  of  the  Toxin. — The  mode  of  preparation  and 
the  conditions  affecting   the  development   of   diphtheria  toxin 
have  already  been  described  (p.  406).     In  the  case  of  tetanus  the 
growth  takes  place  in  glucose  bouillon  under  an  atmosphere  of 
hydrogen  (vide  p.  66).     In  either  case  the  culture   is  filtered 
through  a  Chamberland  filter  when   the   maximum   degree   of 
toxicity  has  been  reached.     The  term  "  toxin  "  is  usually  applied 
for  convenience  to  the  filtered  (i.e.  bacterium-free)  culture. 

2.  Estimation   of  the   Toxin. — The    power   of   the   toxin    is 
estimated  by  the  subcutaneous  injection  of  varying  amounts  in 
a   number  of  guinea-pigs,  and  the   minimum   dose   which  will 
produce  death  is  thus  obtained.     This,  of  course,  varies  in  pro- 
portion to  the  weight  of  the  animal,  and  is  expressed  accordingly. 
In  the  case  of  diphtheria,  in  Ehrlich's  standard,  the  minimum 
lethal  dose — known  as  M.L.D. — is  the  smallest  amount  which 
will  certainly  cause  death  in  a  guinea-pig  of  250  grms.  within 
four  days.     The  testing  of  a  toxin  directly  is  a  tedious  process, 
and  in  actual  practice,  where  many  toxins  have  to  be  dealt  with, 
it  is  found  more  convenient  to  test  them  by  finding  how  much 
will  be  neutralised  by  a  certain  amount  of  a  standard  antitoxic 
serum,  namely,  an  "  immunity  unit "  (p.  524). 

3.  Development  of  Antitoxin. — The   earlier   experiments    on 
tetanus  and  diphtheria  were  performed  on  small  animals,  such 
as  guinea-pigs,  but  afterwards  the  sheep  and  the  goat  were  used, 
and  finally  horses.     In  the  case  of  the  small  animals  it  was 
found  advisable  to  use  in  the  first  stages  of  the  process  either  a 
weak  toxin  or  a  powerful  toxin  modified  by  certain  methods. 
Sn.-h   lilt-thuds  are  the  addition  to  the  toxin  of  terchloride    of 
in. line  (Behring   and  Kitasato),  the  addition  of  Gram's  iodine 
solution  in  the  proportion  of  one  to  three  (Roux  and  Vaillard), 
ami   the  plan,  adopted  by  Vaillard    in  the  case  of  tetanus,  of 
usini:  a  >rri«'>  of  toxins  weakened  to  varying  degrees  by  being  ex- 
posed to  different  temperatures,  namely,  60°,  and  55°,  and  50°  C. 
In  the  case  of  large  animals  immunisation  is  sometimes  started 


524  IMMUNITY 

with  small  doses  of  unaltered  toxin ;  and  the  doses  are  gradually 
increased.  The  toxin  is  at  first  injected  into  the  subcutaneous 
tissues,  later  into  a  vein.  Ultimately  300  c.c.,  or  more,  of  active 
diphtheria  toxin  thus  injected  may  be  borne  by  a  horse,  such  a 
degree  of  resistance  being  developed  after  the  treatment  has  been 
carried  out  for  two  or  three  months.  The  antitoxin  content  of 
the  serum  is  estimated  from  time  to  time,  the  object  being,  of 
course,  to  raise  it  to  as  high  a  figure  as  possible.  It  is  found 
that  each  injection  produces  a  certain  amount  of  fall  in  the  anti- 
toxin value,  and  this,  in  favourable  cases,  is  followed  by  a  rise  to 
a  higher,  level  than  before,  the  former  event  being  due  in  part 
to  the  combination  of  a  portion  of  the  antitoxin  with  the  toxin 
introduced.  (Similar  phenomena  are  observed  in  the  develop- 
ment of  all  other  classes  of  anti-substances.)  In  all  cases  of 
immunising  the  general  health  of  the  animal  ought  not  to  suffer. 
If  the  process  is  pushed  too  rapidly  the  antitoxic  power  of  the 
serum  may  diminish  instead  of  increasing,  and  a  condition  of 
marasmus  may  set  in  and  may  even  lead  to  the  death  of  the 
animal.  After  a  sufficiently  high  degree  of  antitoxic  power 
has  been  reached,  the  animal  is  bled  under  aseptic  pre- 
cautions, and  the  serum  is  allowed  to  separate  in  the  usual 
manner.  It  is  then  ready  for  use,  but  some  weak  antiseptic, 
such  as  '5  per  cent  carbolic  acid,  is  usually  added  to  prevent  its 
decomposing.  Other  antitoxic  sera  are  prepared  in  a  correspond- 
ing manner.  Some  further  facts  about  antitetanic  serum  are 
given  on  p.  429.  (In  immunisation  of  small  animals  an  indica- 
tion of  their  general  condition  may  be  obtained  by  weighing 
them  from  time  to  time.) 

4.  Estimating  the  Antitoxic  Power  q/,  or  "standardising"  the 
Serum. — This  is  done  by  testing  the  effect  of  various  quantities 
of  the  serum  of  the  immunised  animal  against  a  certain  amount 
of  toxin.  Various  standards  have  been  used,  of  which  the  two 
chief  are  that  of  Ehrlich  and  that  of  Roux.  Ehrlich  has  adopted 
as  the  immunity  unit  the  amount  of  antitoxic  serum  which  will 
neutralise  100  times  the  minimum  lethal  dose  of  toxin,  serum 
and  toxin  being  mixed  together,  diluted  up  to  4  c.c.  and  injected 
subcutaneously  into  a  guinea-pig  of  250  grms.  weight,  the 
prevention  of  the  death  of  the  animal  within  four  days  being 
taken  as  the  indication  of  neutralisation.  1  c.c.  of  a  serum,  of 
which  '02  c.c.  will  protect  against  a  hundred  times  the  lethal 
dose,  will  possess  50  immunity  units,  and  20  c.c.  of  this  serum 
1000  imm unity  units.  Sera  have  been  prepared  of  which  1  c.c. 
has  the  value  of  800  units  or  even  more.  As  a  standard  in 
testing,  Ehrlich  employs  quantities  of  serum  of  known  antitoxic 


USE  OF  ANTITOXIC  SERA  525 

power  in  a  dry  condition,  preserved  in  a  vacuum  in  a  cool  place, 
and  in  the  absence  of  light.  A  thoroughly  dry  condition  is 
ensured  by  having  the  glass  bulb  containing  the  dried  serum 
connected  \\ith  another  bulb  containing  anhydrous  phosphoric 
acid.  With  such  a  standard  test-serum  any  newly  prepared 
serum  can  readily  be  compared. 

Roux  has  adopted  a  standard  which  represents  the  animal  weight  in 
grammes  protected  by  1  c.c.  of  serum  against  the  dose  of  virulent  bacilli 
lethal  to  a  control  guinea-pig  in  thirty  hours,  the  serum  being  injected 
twelve  hours  previously.  Thus,  if  '01  c.c.  of  a  serum  will  protect  a 
guinea-pig  of  500  grms.  against  the  lethal  dose,  1  c.c.  (1  grin.)  will  pro- 
tect 50,000  grms.  of  guinea-pig,  and  the  value  of  the  serum  will  be  50,000. 

Use  of  Antitoxic  Sera. — In  all  cases  the  antitoxic  serum  ought 
to  be  injected  as  early  in  the  disease  as  possible,  and  in  large 
doses.  In  the  case  of  diphtheria  1500.  immunity  units  of  anti- 
toxic serum  was  the  amount  first  recommended  for  the  treatment 
of  a  bad  case,  but  the  advisability  of  using  larger  doses  has 
gradually  become  more  and  more  evident.  Sidney  Martin 
recommends  that  as  much  as  4000  units  should  be  administered 
at  once,  and  that  if  necessary  this  quantity  should  be  repeated. 
A  strong  serum  prepared  by  Behring  contains  3000  units  in 
5  to  6  c.c.,  but  even  stronger  sera  may  be  obtained.  Even  very 
large  doses  of  antitoxic  serum  are  without  any  harmful  effects 
beyond  the  occasional  production  of  urticarial  and  erythematous 
rashes  (p.  563).  Where  large  quantities  of  serum  require  to  be 
administered,  as  is  always  the  case  with  antitetanic  serum,  in- 
jections must  be  made  at  different  parts  of  the  body ;  preferably 
not  more  than  20  c.c.  should  be  injected  at  one  place.  In  recent 
times  intravenous  injection  has  been  introduced,  the  advantage 
being  greater  rapidity  of  action.  The  immunity  conferred  by 
injection  of  antitoxic  serum  lasts  a  comparatively  short  time, 
usually  a  few  weeks  at  longest. 

Sera  of  Animals  immunised  against  Vegetable  and  Animal 
Poisons. — It  was  found  by  Ehrlich  in  the  case  of  the  vegetable 
toxins,  ricin  and  abrin,  and  also  by  Calmette  and  Fraser  in  the 
case  of  the  snake  poisons,  that  the  serum  of  animals  immunised 
against  these  respective  substances  had  a  protective  effect  when 
injected  along  with  them  into  other  animals.  Ehrlich  found, 
for  example,  that  the  serum  of  a  mouse  which  had  been  highly 
immunised  against  ricin  by  feeding  as  described  above,  could 
protect  another  mouse  against  forty  times  the  fatal  dose  of  that 
substance.  He  considered  that  in  the  case  of  the  two  poisons, 
antagonistic  substances — "anti-ricin"  and  "  anti-abrin  "—  were 


526  IMMUNITY 

developed  in  the  blood  of  the  highly  immunised  animals.  A 
corresponding  antagonistic  body,  to  which  Eraser  gave  the  name 
"  antivenin,"  appears  in  the  blood  of  animals  in  the  process  of 
immunisation  against  snake  poison. 

These  investigations  are  specially  instructive,  as  such  vegetable 
and  animal  poisons,  both  as  regards  their  local  action  and  the 
general  toxic  phenomena  produced  by  them,  present,  as  we  have 
seen,  an  analogy  to  various  toxins  of  bacteria. 

Nature  of  Antitoxic  Action. — This  subject  is  only  part  of  the 
general  question  with  regard  to  the  relation  of  anti-substances 
to  their  corresponding  antigens,  but  it  is  with  regard  to  anti- 
toxic action  that  most  of  the  work  has  been  done.  We  have  to 
consider  here  two  points,  namely,  (a)  the  relation  of  antitoxin 
to  toxin,  and  (b)  the  source  of  the  antitoxin.  With  regard  to  the 
former  subject  there  is  now  no  doubt  that  the  antagonism 
between  toxin  and  antitoxin  is  not  a  physiological  one  but  that 
the  two  bodies  unite  in  vitro  to  form  a  compound  inert  towards 
the  living  tissues,  there  being  in  the  toxin  molecule  an  atom 
group  which  has  a  specific  affinity  for  the  antitoxin  molecule  or 
part  of  it.  We  shall  consider  the  facts  in  favour  of  this  view, 
and  in  doing  so  we  must  also  take  into  account  the  anti-sera  of 
the  vegetable  toxins,  of  snake  poisons,  etc. 

When  toxin  and  antitoxin  are  brought  together  in  vitro,  it 
can  be  proved  that  their  behaviour  towards  each  other  resembles 
what  is  observed  in  chemical  union.  Thus  it  has  been  found 
that  a  definite  period  of  time  elapses  before  the  neutralisation 
of  the  toxin  is  complete,  that  neutralisation  takes  place  more 
rapidly  in  strong  solutions  than  in  weak,  and  that  it  is  hastened 
by  warmth  and  delayed  by  cold.  C.  J.  Martin  and  Cherry,  and 
also  Brodie,  have  shown,  that  in  the  case  of  diphtheria  toxin  and 
in  that  of  an  Australian  snake  poison  the  toxin  molecules  will 
pass  through  a  colloid  membrane  (p.  193),  whilst  those  of  the 
corresponding  antitoxin  will  not.  Now,  if  a  mixture  of  equivalent 
parts  of  toxin  and  antitoxin  is  freshly  prepared  and  at  once 
filtered,  a  certain  amount  of  toxin  will  pass  through,  but  the 
longer  such  a  mixture  is  allowed  to  stand  before  filtration  the 
less  toxin  passes,  till  a  time  is  reached  when  no  toxin  is  found 
in  the  filtrate.  Further,  if  the  portion  of  fluid  which  at  this 
stage  has  not  passed  through  the  filter  be  injected  into  an  animal 
no  symptoms  take  place ;  this  shows  that  after  a  time  neutral- 
isation is  complete.  Again,  in  cases  where  the  toxin  has  some 
definite  physical  effect,  demonstrable  in  vitro,  e.g.  lysis,  aggluti- 
nation, coagulation,  or  the  prevention  of  coagulation,  its  action 
can  be  annulled  by  the  antitoxin ;  in  such  circumstances 


NATURE  OF  ANTITOXIC  ACTION  527 

manifestly  no  physiological  action  of  antitoxin  through  the 
medium  of  the  cells  of  the  body  can  come  into  play.  These 
facts  are  practically  conclusive  in  favour  of  antitoxin  action 
depending  upon  a  direct  union  of  the  two  substances  concerned, 
and  Morgenroth  has  recently  shown  that  the  combination  toxin- 
antitoxin  can  be  broken  up  by  the 'action  of  hydrochloric  acid 
and  the  two  constituents  recovered. 

Although  authorities  are  now  agreed  as  to  the  direct  com- 
bination of  toxin  and  antitoxin,  there  is  still  much  uncer- 
tainty as  to  the  exact  nature  of  this  union.  Regarding  this 
subject  there  may  be  said  to  be  three  chief  views — (a) 
that  of  Ehrlich,  according  to  which  there  is  a  firm  chemical 
union  of  toxin  and  antitoxin,  and  the  former  is  not  homo- 
geneous but  has  a  complex  structure ;  (6)  that  of  Arrhenius  and 
Madsen,  who  consider  that  the  phenomena  correspond  to  the 
behaviour  of  two  substances  in  weak  chemical  union ;  and  (c)  that 
of  Bordet,  who  regards  the.  combination  not  to  be  of  chemical, 
but  of  physical  nature,  corresponding  to  a  process  of  adsorption. 
Controversy  on  this  question  may  be  said  to  date  from  the 
important  work  of  Ehrlich  on  the  neutralisation  of  diphtheria 
toxin.  Using  an  immunity  unit  of  antitoxin  (the  equivalent  of 
100  doses  of  toxin)  he  determined  with  any  example  of  crude 
toxin  the  largest  amount  of  toxin  which  could  be  neutralised 
completely,  so  that  no  symptoms  resulted  from  an  injection  of 
the  mixture.  This  amount  he  called  the  limes  null  dose,  ex- 
pressed as  L0.  He  then  investigated  the  effects  of  adding  larger 
amounts  of  toxin  to  the  immunity  unit  and  observed  the 
quantity  which  was  first  sufficient  to  produce  a  fatal  result, 
that  is,  which  contained  one  M.L.D.  of  free  toxin;  this  amount 
he  called  the  limes  todtlich,  fatal  limit,  expressed  as  Lt.  Now 
if,  as  he  supposed,  the  union  of  toxin  and  antitoxin  resembled 
that  of  a  strong  acid  and  base,  Lt  -  L0  ought  to  be  the  equiva- 
lent of  a  minimum  lethal  dose  of  the  toxin  alone.  This,  how- 
ever, was  never  found  to  be  the  case,  the  difference  being  always 
considerably  more  than  one  M.L.D.  For  example,  in  the  case 
of  one  toxin,  M.L.D.  =  '0165  c.c.,  Lt=l'26  c.c.,  L0=-9  c.c. ; 
difference  = -36  c.c.,  i.e.  21 '9  M.L.D.  This,  in  brief,  is  what 
is  known  as  the  "Ehrlich  phenomenon,"  and  it  has  been 
explained  by  him  as  the  result  of  the  presence  of  toxoids  (vide 
p.  198),  i.e.  toxin  molecules  in  which  the  toxophorous  group  has 
become  degenerated.  He  distinguishes  three  possible  varieties 
of  such  bodies  according  to  the  affinity  of  the  haptophorous 
group,  namely,  prototoxoid  with  more  powerful  affinity  than  the 
toxin  molecule,  epitoxoid  with  less  powerful  affinity,  and  syntoxoid 


528  IMMUNITY 

with  equal  affinity.  The  presence  of  epitoxoids  would  manifestly 
explain  the  above  phenomenon.  The  L0  dose  would  represent 
toxin  +  epitoxoid  molecules  all  united  to  antitoxin  molecules  and 
the  addition  of  another  M.L.D.  of  toxin  would  not  result  in 
there  being  a  free  fatal  dose,  but  in  the  added  toxin  taking  the 
place  of  epitoxoid.  Several  lethal  doses  would  need  to  be  added 
before  the  mixture  was  sufficient  to  produce  a  fatal  result ;  that  is, 
Lt  — L0  would  equal  several  M.L.D.s.  Ehrlich  observed  another 
fact  strongly  in  favour  of  the  existence  of  toxoids,  namely,  that 
in  the  course  of  time  the  toxin  might  become  much  weakened, 
so  that  in  one  case  observed  the  M.L.D.  came  to  be  three  times 
the  original  fatal  dose,  and  still  the  amount  of  antitoxin  neces- 
sary to  neutralise  it  completely  was  the  same  as  before.  Ehrlich 
also  investigated  the  effects  of  partial  neutralisation  of  the  L0 
amount  of  toxin,  that  is,  he  added  to  this  amount  different 
fractions  of  an  immunity  unit  and  estimated  the  toxicity  of  the 
mixture.  He  found  by  this  method  that  the  neutralisation  of 
the  toxin  did  not  take  place  gradually,  but  as  if  there  were  dis- 
tinct bodies  present  with  different  combining  affinities — the 
graphic  representation  of  the  effects  of  the  mixture  not  being  a 
curve  but  a  step-stair  line.  Thus  he  distinguished  proto-,  deutero-, 
and  trito-toxins  (with  corresponding  toxoids).  It  will  thus  be 
seen  that  Ehrlich  regards  the  combination  toxin-antitoxin  to  be 
a  firm  one,  and  that  the  neutralisation  phenomena  are  to  be 
explained  by  the  complicated  constitution  of  the  crude  toxin. 

The  chief  criticism  of  Ehrlich's  views  has  come  from  the 
important  work  of  Madsen  and  Arrhenius.  Their  main  con- 
tention is  that  the  toxin-antitoxin  combination  is  not  a  firm  one 
but  a  reversible  one,  and  is  governed  by  the  laws  of  physical 
chemistry.  For  example,  in  the  case  of  a  mixture  of  ammonia 
and  boracic  acid  (i.e.  of  a  weak  base  and  a  weak  acid) 
in  solution,  there  is  a  constant  relation  between  the  amounts 
of  each  of  the  substances  in  the  free  condition  and  the 
amounts  in  combination, — the  combination  is  reversible,  so  that 
if  some  of  the  free  ammonia  were  removed  a  certain  amount  of 
the  combined  ammonia  would  become  dissociated  to  take  its 
place ;  further,  if  to  the  mixture,  in  a  state  of  equilibrium, 
more  ammonia  or  more  boracic  acid  were  added,  part  would 
remain  free  while  part  would  combine.  Accordingly,  if  toxin 
and  antitoxin  behaved  in  a  similar  manner  an  explanation  of  the 
Ehrlich  phenomenon  would  be  afforded.  Madsen  and  Arrhenius 
have  worked  out  the  question  in  the  case  of  a  great  many  toxins, 
and  find  that  the  graphic  representation  of  neutralisation  is  in 
every  case  a  curve  which  can  be  represented  by  a  formula.  It 


MODE  OF  PRODUCTION  OF  ANTITOXINS     529 

should  be  noted  in  connection  with  this  controversy  that  there  are 
two  questions  which  may  be  independent  of  each  other,  namely, 
(1)  does  the  "toxin"  in  any  particular  case  represent  a  single 
substance  or  several1?  (2)  What  is  the  nature  of  the  combination 
of  any  one  constituent  substance  and  its  anti-substance — is  it 
reversible  or  is  it  not  ?  It  may  be  said  that  it  is  practically 
impossible  to  explain,  the  facts  with  regard  to  diphtheria  toxin 
on  the  hypothesis  of  a  single  substance,  even  if  this  should  have 
its  combining  and  toxic  actions  equally  weakened  ;  "  toxoids  "  in 
Ehrlich's  sense  must  in  our  opinion  be  supposed.  Then  there  is 
an  important  fact  established  by  Danysz  and  by  v.  Dungern, 
namely  that  the  amount  of  toxin  neutralisable  by  a  given  amount 
of  antitoxin  is  different  according  as  the  toxin  is  added  in  several 
moieties  or  all  at  once — in  the  latter  case  the  amount  of  toxin 
neutralisable  is  greater.  There  seems  no  explanation  of  this 
according  to  the  view  of  Madsen  and  Arrhenius,  as  the  same  state 
of  equilibrium  ought  to  be  reached  in  the  two  cases,  that  is,  the 
amounts  of  toxin  neutralised  should  be  the  same. 

An  important  t'ai-tnr  in  the  union  of  toxin  and  antitoxin  is 
the  time  necessary  for  the  union  to  be  complete.  Morgenroth 
lias  shown  that  in  the  case  of  diphtheria  toxin  this  is  considerable, 
— about  twenty-four  hours.  Up  to  this  time,  mixtures  of  toxin 
and  antitoxin,  \\hrn  injected  intravenously,  show  decreasing 
degrees  of  toxicity  according  to  the  time  they  have  kept.  On 
the  other  hand,  when  the  subcutaneous  method  of  injection  is 
used  the  time  interval  has  no  effect,  and  this  he  considers  to  be 
due  to  a  catalytic  action  of  the  tissues  which  accelerates  the 
union  of  the  two  substances.  A  striking  phenomenon,  which 
apparently  points  to  the  reversibility  of  the  combination,  was 
noted  by  Behring  in  the  case  of  diphtheria  toxin,  and  afterwards 
studied  l>y  Madsen  and  by  Otto  and  Sachs  in  the  case  of 
botulismus  toxin,  namely,  that  when  a  mixture  of  toxin  and 
antitoxin  was  found  to  be  neutral  on  injection,  a  fraction  of  the 
mixture  might  produce  toxic  phenomena  or  even  death.  This 
wa«*  apparently  due  to  dissociation  of  the  toxin  in  the  greater 
dilution,  and  in  favour  of  this  being  the  case  Otto  and  Sachs 
found  that  when  the  mixture  was  allowed  to  stand  for  twenty- 
ton!1  hours,  so  that  combination  was  complete,  the  phenomenon 
no  longer  occurred.  It  was  shown  by  Morgenroth  and  by  Muir 
independently  that  the  union  of  a  hasinolytic  immune-body  with 
tin-  ••onvspondinu;  red  corpuscle  was  of  reversible  nature,  and 
tin-  latter  observer  found  that  in  this  case  the  union  was  not 
increased  in  iirnmess  after  twenty-four  hours.  There  is  little 
doubt  that  there  are  varying  degrees  of  firmness  of  union  of  an 

34 


530  IMMUNITY 

antigen  and  its  anti-substance  and  varying  periods  necessary  for 
the  combination  to  become  complete  ;  and  it  is  quite  evident  that 
if  there  should  be  several  toxic  bodies  in  a  "  toxin,"  and  that  if 
the  union  of  some  of  these  with  antitoxin  should  be  reversible, 
the  problem  becomes  one  of  extreme  complexity. 

There  has  recently  been  a  tendency  on  the  part  of  some 
authorities  to  consider  that  the  union  of  toxin-antitoxin  does  not 
correspond  to  what  takes  place  in  ordinary  chemical  union,  but 
is  a  physical  interaction  of  bodies  in  a  colloidal  state,  the  action 
being  one  of  the  so-called  adsorption  phenomena.  The  smaller 
toxin  molecule  becomes  entangled,  as  it  were,  in  the  larger 
antitoxin  one,  very  much  as  a  dye  becomes  attached  to  the 
structure  of  a  thread.  Bordet  has  long  maintained  a  theory  of 
this  nature,  and  gives  reasons  for  believing  that  there  is  no 
definite  quantitative  relationship  in  the  combination  of  the 
molecules  of  the  two  substances,  different  amounts  of  antitoxin 
affecting  in  varying  degree  all  the  molecules  of  a  given  amount 
of  toxin.  A  statement  on  the  general  question  is  at  present 
impossible ;  we  can  only  say  that  direct  combination  of  the  two 
bodies  does  occur;  that  sometimes,  probably  often,  the  "toxin" 
contains  different  toxic  bodies  with  varying  affinity ;  and  that 
in  a  few  instances  the  combination  has  been  proved  to  be  revers- 
ible, but  to  what  extent  this  is  generally  true  remains  still  to  be 
determined. 

The  next  question  to  be  considered  is  the  source  of  antitoxin. 
The  following  three  possibilities  present  themselves  :  (a)  antitoxin 
may  be  formed  from  the  toxin,  i.e.  may  be  a  "  modified  toxin  "  ; 
(b)  antitoxin  may  be  the  result  of  an  increased  formation  of 
molecules  normally  present  in  the  tissues ;  (c)  antitoxin  may  be 
an  entirely  new  product  of  the  cells  of  the  body.  It  can  now  be 
stated  that  antitoxin  is  not  a  modified  toxin.  It  has  been  shown, 
for  example,  that  the  amount  of  antitoxin  produced  by  an  animal 
may  be  many  times  greater  than  the  equivalent  of  toxin  injected  ; 
and  further,  that  when  an  animal  is  bled  the  total  amount  of 
antitoxin  in  the  blood  may  some  time  afterwards  be  greater 
than  it  was  immediately  after  the  bleeding,  even  although  no 
additional  toxin  is  introduced.  This  latter  circumstance 
shows  that  antitoxin  is  formed  by  the  cells  of  the  body.  If 
antitoxin  is  a  product  of  the  cells  of  the  body,  we  are  almost  com- 
pelled, on  theoretical  grounds,  to  conclude  that  it  is  not  a  newly- 
manufactured  substance,  but  a  normal  constituent  of  the  living 
cells  which  is  produced  in  increased  quantity.  We  have,  how- 
ever, direct  evidence  of  the  presence  of  antitoxin  under  normal  con- 
ditions,— the  presence  of  such  being  shown  by  its  uniting  with 


MODE  OF  PRODUCTION  OF  ANTITOXINS      531 

toxin  and  rendering  it  inert.  Normal  horse  serum,  to  mention 
an  example,  may  have  a  varying  amount  of  antitoxic  action  to 
the  diphtheria  poison,  ox-bile  has  a  similar  action  to  snake  poison, 
whilst  in  the  case  of  other  anti-substances — such  as  agglutinins, 
bacteriolysins,  haemolysins,  etc. — whose  production  is  governed 
by  the  same  laws,  numerous  examples  might  be  given.  It  is, 
however,  rather  to  the  protoplasm  of  living  cells  than  to  the  serum 
that  we  must  look  for  the  source  of  antitoxins.  In  the  first  place, 
we  have  evidence  that  in  the  living  body  bacterial  toxins  enter 
into  combination  with,  or,  as  it  is  often  expressed,  are  fixed  by 
the  tissues — presumably  by  means  of  certain  combining  affinities. 
This  has  been  shown  by  the  experiments  of  Donitz  and  of 
Heymans  with  tetanus  toxin.  We  have,  in  such  cases,  however, 
no  evidence  as  to  where  the  toxin  is  fixed  beyond  that  supplied 
l>y  the  occurrence  of  symptoms.  Another  line  of  research  which 
has  been  followed  is  to  bring  emulsions  of  various  organs  into 
contact  with  a  given  toxin  and  observe  whether  any  of  the 
toxicity  is  removed.  This  was  first  carried  out  by  Wassermann 
and  Takaki,  who  investigated  the  action  of  emulsions  of  the 
central  nervous  system  of  the  susceptible  guinea-pig  on  tetanus 
toxin.  They  found  in  this  way  that  the  nervous  system  con- 
tained bodies  which  had  a  neutralising  effect  on  the  toxin. 
For  example,  it  was  shown  that  1  c.c.  of  emulsion  of  brain  and 
spinal  cord  was  capable  of  protecting  a  mouse  against  ten  times 
the  fatal  dose  of  toxin.  These  observations  have  been  confirmed, 
though  their  significance  has  been  variously  interpreted  :  and  in 
view  of  recently  ascertained  facts  with  regard  to  processes  of 
physical  adsorption,  it  is  quite  possible  that  this  neutralisation 
of  toxin  does  not  represent  a  specific  union  as  in  the  case  of 
antitoxin  action.  We  may  note,  however,  that  it  is  not  a  serious 
objection  that  in  certain  animals  other  tissues  than  that  of  the 
central  nervous  system  can  combine  with  tetanus  toxin — this 
might  take  place  with  or  without  resulting  symptoms. 

It  will  be  seen  from  what  has  been  stated  with  regard  to  the 
relation  of  toxin  and  antitoxin,  that  the  fixation  of  toxin  by  the 
tissues  leads  up  theoretically  to  the  possible  production  of  anti- 
toxin. In  other  words,  the  substance  which,  when  forming  part 
of  the  cells,  fixes  the  toxin  and  thus  serves  as  the  means  of 
jmisDiiiiitr,  may  act  as  an  antitoxin  when  free  in  the  blood. 
This  will  be  discussed  below  in  connection  with  Ehrlich's  theory 
of  jussive  immunity.  We  may  conclude  by  saying  that  anti- 
tai-in  is  2)?obably  represented  by  molecules  normally  present  in 
the  cells  or  (more  rarely)  in  tJie  fluids  of  the  body. 

Of  the  chemical  nature  of  antitoxins  we  know  little.     From 


532  IMMUNITY 

their  experiments  C.  J.  Martin  and  Cherry  deduced  that  while 
toxins  are  probably  of  the  nature  of  albumoses,  the  antitoxins 
probably  have  a  molecule  of  greater  size,  and  may  be  allied  to 
the  globulins.  Such  a  supposed  difference  in  the  sizes  of  the 
molecules  might  explain  the  fact,  observed  by  Fraser  and  also 
by  C.  J.  Martin,  that  antitoxin  is  much  more  slowly  absorbed 
when  introduced  subcutaneously  than  is  the  case  with  toxin. 
Hiss  and  Atkinson  also  came  to  the  conclusion  that  antitoxin 
belongs  to  the  globulins.  They  found  that  the  precipitate  with 
magnesium  sulphate  from  anti-diphtheria  serum  contained 
practically  all  the  antitoxin,  and  that  any  substance  obtained 
which  had  an  antitoxic  value  gave  all  the  reactions  of  a  globulin  ; 
and  this  result  has  been  confirmed  by  others.  They  also  found 
that  the  percentage  amount  of  globulin  precipitated  from  the 
serum  of  the  horse  increased  after  it  was  treated  in  the  usual 
way  for  the  production  of  antitoxin.  Ledingham  observed  an 
increase  of  globulin  during  the  process  of  immunisation  of  a 
horse  which  yielded  a  high-grade  antitoxic  serum,  and  he  ascer- 
tained that  while  this  increase  was  more  on  the  part  of  the 
euglobulin  than  of  the  pseudoglobulin  fraction,  most  of  the 
antitoxin  was  contained  in  the  latter. 

Antitoxin,  when  present  in  the  serum,  leaves  the  body  by  the 
various  secretions,  and  in  these  it  has  been  found,  though  in 
much  less  concentration  than  in  the  blood.  It  is  present  in  the 
milk,  and  a  certain  degree  of  immunity  can  be  conferred  on 
animals  by  feeding  them  with  such  milk,  as  has  been  shown  by 
Ehrlich,  Klemperer,  and  others.  Klemperer  also  found  traces  of 
antitoxin  in  the  yolk  of  eggs  of  hens  whose  serum  contained 
antitoxin.  Bulloch  also  found  in  the  case  of  hseinolytic  sera 
(vide  infra)  that  the  anti-substance  ("immune-body  ")  is  trans- 
mitted from  the  mother  to  the  offspring. 

Antibacterial  Serum. — The  stages  in  the  preparation  of 
antibacterial  sera  correspond  to  those  in  the  case  of  antito'xic 
sera,  but  living,  or,  in  the  early  stages,  dead  cultures  are  used 
instead  of  toxin  separated  by  filtration,  and  in  order  to  obtain  a 
serum  of  high  antibacterial  power  a  very  virulent  culture  in  large 
doses  must  be  ultimately  tolerated  by  the  animal.  For  this 
purpose  a  fairly  virulent  culture  is  obtained  fresh  from  a  case  of 
the  particular  disease,  and  its  virulence  may  be  further  increased 
by  the  method  of  passage.  This  method  of  obtaining  a  high' 
degree  of  immunity  against  the  microbe  is  specially  applicable 
in  the  case  of  those  organisms  which  invade  the  tissues  and 
multiply  to  a  great  extent  within  the  body,  and  of  which  the 
toxic  effects,  though  always  existent,  are  proportionately  small 


PROPERTIES  OF  ANTIBACTERIAL  SERUM      533 

in  relation  to  the  number  of  organisms  present.  The  method 
has  been  applk-d  in  the  case  of  the  typhoid  and  cholera  organ- 
isms, the  bacillus  of  bubonic  plague,  the  bacillus  coli  communis, 
the  pneumococcus,  streptococcus  (Marmorek),  and  many  others. 
In  fact,  it  seems  capable  of  very  general  application. 

The  important  result  obtained  by  such  experiments  is,  that  if 
an  animal  be  highly  immunised  by  the  method  mentioned,  the 
development  of  the  immunity  is  accompanied  by  the  appearance 
in  the  blood  of  protective  substances,  which  can  be  transferred  to 
another  animal.  The  law  enunciated  by  Behring  regarding 
immunity  against  toxins  thus  holds  good  in  the  case  of  the 
living  organisms,  as  was  first  shown  by  Pfeiffer.  The  latter 
found,  for  example,  that  in  the  case  of  the  cholera  organisms,  so 
high  a  degree  of  immunity  could  be  produced  in  the  guinea-pig, 
that  '002  c.c.  of  its  serum  would  protect  another  guinea-pig 
against  ten  times  the  lethal  dose  of  the  organisms,  when  injected 
along  with  them.  Here  again  is  presented  the  remarkable 
potency  of  the  antagonising  substances  in  the  serum,  which  in 
this  case  lead  to  the  destruction  of  the  corresponding  microbe. 

The  anti-stre2>tococcic  serum  of  Marmorek  may  be  briefly  described,  as 
it  has  come  into  extensive  practical  use.  This  observer  found  that  he 
could  intensify  the  virulence  of  a  streptococcus  by  growing  it  alternately 
in  the  peritoneal  cavity  of  a  guinea-pig  and  in  a  mixture  of  human  blood 
serum  and  bouillon  (vide  p.  41).  The  virulence  became  so  enormously 
increased  by  this  method,  that  when  only  one  or  two  organisms  were 
introduced  into  the  tissues  of  a  rabbit  a  rapidly  fatal  septicaemia  was 
produced.  Streptococci  of  this  high  degree  of  virulence  were  used  first 
by  subcutaneous,  afterwards  by  intravenous  injection,  to  develop  a  high 
degree  of  resistance  in  the  horse.  Injections  were  continued  over  a  con- 
siderable period  of  time,  and  the  protective  power  of  the  serum  was 
tested  by  mixing  it  with  a  certain  dose  of  the  virulent  organisms,  and 
then  injecting  into  a  rabbit.  The  serum  of  a  horse  highly  immunised  in 
this  way  constitutes  the  anti-streptococcic  serum  which  has  been  exten- 
sively used  in  many  cases  of  streptococcic  invasion  in  the  human  subject. 
Marmorek,  however,  found  that  this  serum  had  little  antitoxic  power, 
that  is,  could  only  protect  from  a  comparatively  small  dose  of  toxin 
obtained  by  filtration  of  cultures. 

Anti-typhoid,  anti-cholera,1  anti-pneumococcic,  anti-plague, 
and  other  sera  are  all  prepared  in  an  analogous  manner. 

Properties  of  Antibacterial  Serum. — We  have  here  to 
consider  the  three  main  actions  mentioned  above,  namely, 
(a)  bactericidal  and  lysogenic  action,  (b)  opsonic  action,  and 
(c)  agglutinative  and  the  closely  allied  precipitating  action.  Of 

1  A  true  antitoxic  cholera  serum  has  been  prepared  by  Metchnikoff, 
E.  Iloux,  and  Taurelli-Saliuibeui. 


534  IMMUNITY 

these  the  two  first  are  concerned  with  the  protective  property  of 
an  anti-bacterial  serum. 

(a)  Bactericidal  and  Lysogenic  Action. — Pfeiffer  found  that 
if  certain  organisims,  e.g.  the  cholera  spirillum,  were  injected 
into  the  peritoneal  cavity  of  a  guinea-pig  highly  immunised 
against  these  organisms,  they  lost  their  motility  almost  immedi- 
ately, gradually  became  granular,  swollen,  and  then  disappeared 
in  the  fluid — these  changes  constitute  "Pfeiffer's  phenomenon." 
Further,  he  showed  that  the  same  phenomenon  was  witnessed  if 
a  minute  quantity  of  the  anti-serum  was  added  to  a  certain 
quantity  of  the  corresponding  organisms,  and  the  mixture  injected 
into  the  peritoneal  cavity  of  a  non-treated  animal.  Pfeiffer 
found  that  the  serum  of  convalescent  cholera  patients  gave  the 
same  reaction  as  that  of  immunised  animals.  He  obtained  the 
same  reaction  also  in  the  case  of  the  typhoid  bacillus  and  other 
organisms.  From  his  observations  he  concluded  that  the  reaction 
was  specific,  and  could  be  used  as  a  means  of  distinguishing 
organisms  which  resemble  one  another.  He  accordingly  con- 
sidered that  a  specific  substance  was  developed  in  the  process  of 
immunisation,  and  that  this  was  rendered  actively  bactericidal 
by  the  aid  of  the  living  cells  of  the  body.  It  was  subsequently 
shown,  however,  by  Metchnikoff  and  by  Bordet  that  lysogenesis 
might  occur  outside  the  body  by  the  addition  of  fresh  peritoneal 
fluid  or  normal  serum  to  the  heated  immune-serum.  PfeifFer 
also  found  that  an  anti-serum  heated  to  70°  C.  for  an  hour  pro- 
duced the  reaction  when  injected  with  the  corresponding  organisms 
into  the  peritoneum  of  a  fresh  animal.  The  outcome  of  these 
and  subsequent  researches  is  to  show  that  when  an  animal  is 
immunised  against  a  bacterium  a  substance  appears  in  its  serum 
with  combining  affinity  for  that  particular  organism.  This 
substance,  which  is  generally  known  as  the  immune-body, 
amboceptor  (Ehrlich),  or  substance  sensibilisatrice  (Bordet)  is 
comparatively  stable,  resisting  usually  a  temperature  of  70°  C. 
for  an  hour.  It  cannot  produce  the  destructive  effect  alone,  but 
requires  the  addition  of  a  substance  normally  present  in  the  serum, 
which  is  spoken  of  under  various  names — complement  (Ehrlich), 
alexine  or  cytase  (French  writers).  The  complement  is  relatively 
unstable,  being  rapidly  destroyed  by  a  temperature  of  60°  C., 
and  it  is  not  increased  in  amount  during  the  process  of 
immunisation.  Though  ferment-like  in  its  instability,  it  differs 
from  a  ferment  in  being  fixed  or  used  up  in  definite  quantities. 

Eecent  observations  show  that  complement  is  not  a  single  substance, 
but  is  really  made  up  of  two  components.  Ferrata,  who  was  the  first  to 
establish  this  fact,  employed  the  following  method  :  Fresh  guinea-pig's 


PROPERTIES  OF  ANTIBACTERIAL  SERUM      535 

scrum  is  dialysed  against  running  water  for  twenty-four  hours  ;  the 
precipitate  which  has  formed  at  the  end  of  that  time  is  separated  by  the 
c.'iitrifugp,  washed  several  times  in  distilled  water,  and  then  dissolved  in 
normal  salt  solution.  The  separated  fluid  is  passed  through  thick  filter 
paper.  The  component  in  the  solution  of  the  precipitate  unites  directly 
with  sensitised  corpuscles — and  then  that  in  the  separated  fluid  enters 
into  combination  ;  hence  they  have  been  called  by  Brand  "middle-piece" 
and  "end-piece"  respectively.  The  separation  by  such  a  method  is, 
however,  far  from  being  a  complete  one.  Sachs  and  Altmann  have 
introduced  the  following  method  :  To  *5  c.c.  of  fresh  guinea-pig's  serum 
is  added  4'1  c.c.  of  a  7575—3^  normal  solution  of  hydrochloric  acid  in 
distilled  water.  The  sediment  is  centrifuged  off  after  the  mixture  has 
been  allowed  to  stand  at  room  temperature  for  an  hour,  and,  after  being 
washed,  is  made  up  with  a  suitable  amount  of  distilled  water.  The 
separated  fluid  is  neutralised  and  made  isotonic  with  '4  c.c.  of  a  sV-3V 
normal  soda  solution  containing  10  per  cent,  sodium  chloride. 

The  phenomenon  of  lysogenesis  is,  however,  only  seen  in  the 
case  of  certain  organisms  when  an  animal  is  highly  immunised 
against  them  ;  the  typhoid  and  cholera  group  are  outstanding 
examples.  It  is  also  to  be  noted  that  it  sometimes  is  seen  in  the 
case  of  a  normal  serum  (vide  Natural  Immunity).  In  other 
cases  the  bactericidal  effect  of  a  serum  may  occur  without  the 
rapid  dissolution  characteristic  of  lysogenesis  though  other 
structural  changes  may  be  produced.  In  still  other  instances, 
e.g.  the  anti-sera  to  staphylococci,  streptococci,  plague  bacilli, 
etc.,  a  bactericidal  effect  may  be  wanting ;  nevertheless  it  may 
be  shown  that  an  immune-body  is  developed  in  the  process  of 
immunisation.  This  may  be  done  by  observing  the  increased 
amount  of  complement  which  is  fixed  through  the  medium  of 
the  anti-serum  (immune-body),  sensitised  red  corpuscles  being 
used  as  the-  test  for  the  presence  of  free  complement.  The 
method  is  described  on  pp.  128-131. 

The  all-important  action  of  the  immune-body  is  thus  to  bring 
an  increased  amount  of  complement  into  union  with  bacteria  ; 
whether  death  of  the  bacteria  will  result  or  not  will  depend 
ultimately  on  their  sensitiveness  to  the  action  of  the  particular 
complement. 

It  is  to  be  noted  that  in  the  case  of  a  bactericidal  serum  there 
is  an  optimum  amount  of  immune-body  which  gives  the  greatest 
bactericidal  effect  with  a  given  amount  of  complement.  If  this 
amount  of  immune -body  be  exceeded,  the  bactericidal  action 
becomes  diminished  and  may  be  practically  annulled.  This 
result,  which  is  generally  known  as  the  Neisser-Wechsberg 
phenomenon,  has  been  the  subject  of  much  controversy,  and 
cannot  yet  be  said  to  be  satisfactorily  explained.  It  would 
accordingly  be  out  of  place  to  discuss  here  the  different  views 


536  IMMUNITY 

with  regard  to  it.  (Regarding  some  theoretical  considerations 
as  to  the  therapeutic  applications  of  antibacterial  sera,  vide 
p.  534.) 

The  laws  of  lysogenesis  are,  however,  not  peculiar  to  the 
case  of  solution  of  bacteria  by  the  fluids  of  the  body,  but,  as  has 
been  shown  within  the  last  few  years,  hold  also  in  the  case  of 
other  organised  substances,  red  corpuscles,  leucocytes,  etc.,  when 
these  are  introduced  into  the  tissues  of  an  animal  as  in  a  process 
of  immunisation.  Of  such  sera  the  hsemolytic  have  been  most 
fully  studied,  and,  owing  to  the  delicacy  of  the  reaction  and  the 
ease  with  which  it  can  be  observed,  have  been  the  means  of 
throwing  much  light  on  the  process  of  lysogenesis,  and  thus  on 
one  part  of  the  subject  of  immunity.  A  short  account  of  their 
properties  may  now  be  given. 

Hcemolytic  and  other  Sera. — It  has  been  known  for  some  time 
that  in  some  instances  the  blood  serum  of  one  animal  has,  in 
a  certain  degree,  the  power  of  dissolving  the  red  corpuscles  of 
another  animal  of  different  species ;  in  other  instances,  how- 
ever, this  property  cannot  be  detected.  Bordet  showed  that 
if  one  animal  were  treated  with  repeated  injections  of  the 
corpuscles  of  another  of  different  species,  the  serum  of  the  former 
acquired  a  marked  haemolytic  property  towards  the  corpuscles  of 
the  latter,  the  property  being  demonstrated  when  the  serum  is 
added  to  the  corpuscles.  He  also  found  that  the  hsemolytic 
property  disappeared  when  the  hsemolytic  serum  was  heated  at 
55°  C.,  but,  as  in  the  case  of  a  bacteriolytic  serum,  was  regained 
on  the  subsequent  addition  of  some  serum  from  a  fresh  (i.e.  non- 
treated)  animal.  These  observations  have  been  fully  confirmed 
and  greatly  extended.  Ehrlich  and  Morgenroth  analysed  the 
phenomena  in  question,  and  showed  that  the  specially  developed 
and  heat-resisting  substance,  "  immune-body,"  entered  into  com- 
bination with  the  red  corpuscles  at  a  comparatively  low 
temperature,  namely,  at  0°  C. ;  whereas  complement  does  not 
combine  at  this  temperature.  In  this  way  a  method  is  supplied 
by  which  the  immune-body  can  be  removed  from  a  hamiolytic 
serum  while  the  complement  is  left.  They  came  to  the  con- 
clusion that  immune-body  combined  with  the  complement 
though  the  combination  was  less  firm  and  only  occurred  at  a 
higher  temperature — best  about  37°  C.  They  therefore  consider 
that  the  immune-body  acts  as  a  sort  of  connecting-link  between 
the  red  corpuscle  and  the  complement,  hence  the  term  "  ambo- 
ceptor "  which  Ehrlich  afterwards  applied.  It  may  be  stated, 
however,  that  the  direct  union  of  complement  and  immune-body 
has  not  been  conclusively  demonstrated.  Muir  and  Browning, 


H^EMOLYTIC  AND  OTHER  SERA  537 

for  example,  found  that  when  a  fresh  serum  is  passed  through  a 
Berkefeld  filter,  complement  is  largely  retained  in  the  pores  of 
the  filter,  whereas  immune-body  passes  through  practically 
unchanged  :  and  that  if  a  mixture  of  complement  and  immune- 
body  be  made  and  filtered  at  a  temperature  of  37°  C.,  the 
amount  of  immune-body  which  passes  through  is  not  diminished, 
whereas  it  would  be  if  it  had  united  with  the  retained  comple- 
ment. Accordingly  by  this  method  there  was  obtained  no 
evidence  of  the  direct  union  of  immune-body  and  comple- 
ment. Bordet  holds  that  the  immune-body  acts  merely 
as  a  sensitising  agent — hence  the  term  substance  sensibili- 
satrice — and  allows  the  ferment-like  complement  to  act.  It 
is  quite  evident  from  his  writings,  however,  that  he  does  not 
mean,  as  is  often  assumed,  that  the  immune-body  causes  some 
lesion  in  the  corpuscle  which  allows  the  complement  to  act,  but 
simply  that  it  produces  in  the  molecules  (receptors)  of  the  red 
corpuscles  an  avidity  for  complement.  All  that  we  can  say 
definitely  at  present  is  that  the  combination  of  receptor  -f 
immune-body  takes  up  complement  in  firm  union  while  neither 
does  so  alone ;  whether  the  immune-body  acts  as  a  link  be- 
tween the  two  or  not  must  be  left  an  open  question.  Even 
after  the  corpuscles  are  laked  with  water  the  receptors  are  not 
destroyed.  Muir  and  Ferguson  have  shown  that  they  can  still 
take  up  immune-body  and,  through  its  medium,  complement, 
just  as  the  intact  corpuscles  do.  Ehrlich  and  Morgenroth  showed 
that  in  some  cases  the  red  corpuscles  can  take  up  much  more 
immune-body  than  is  necessary  for  their  lysis,  and  Muir  found 
in  one  case  studied,  that  each  further  dose  of  immune-body  led 
to  the  fixation  of  more  complement,  so  that  as  many  as  ten 
times  the  htemolytic  dose  of  complement  might  thus  be  used  up. 
It  is  a  matter  of  considerable  importance  that  the  union  of 
immune-body  and  red  corpuscles  can  be  shown  to  be  a  reversible 
action.  If,  as  was  found  by  Morgenroth  and  Muir  indepen- 
dently, corpuscles  treated  with  several  doses  of  immune-body 
and  then  repeatedly  washed  in  salt  solution  be  mixed  with 
untreated  corpuscles  and  allowed  to  remain  for  an  hour,  then 
sufficient  immune-body  will  pass  from  the  former  to  the  latter, 
so  that  all  become  lysed  on  the  addition  of  sufficient  complement. 
The  combination  of  complement,  on  the  other  hand,  is  usually 
of  very  firm  nature.  It  has  been  a  disputed  point  whether  there 
are  several  distinct  complements  in  a  normal  serum  with 
different  relations  to  different  immune-bodies,  for  which  Ehrlich 
and  his  co-workers  have  brought  forward  a  certain  amount  of 
evidence,  or  whether,  as  Bordet  holds,  there  is  a  single  comple- 


538  IMMUNITY 

ment  which  may,  however,  show  slight  variations  in  behaviour 
towards  different  immune-bodies.  There  is  at  least  no  doubt 
that  all  the  complement  molecules  in  a  serum  are  not  the  same. 
For  example,  Muir  and  Browning  have  shown  that  the  treat- 
ment of  a  normal  serum  with  a  small  amount  of  emulsion  of  a 
bacterium  will  remove  the  bactericidal  action  for  another 
bacterium,  whereas  the  amount  of  complement  as  tested  by 
haemolysis  is  practically  unchanged.  They  accordingly  con- 
sider that  there  is  a  moiety  of  complement,  "  bacteriophilic 
complement,"  which  is  specially  concerned  in  bactericidal  action. 
On  the  other  hand,  many  of  the  arguments  adduced  by  Ehrlich 
and  his  co-workers  in  favour  of  a  multiplicity  of  complements 
are  open  to  another  interpretation ;  the  truth  probably  lies 
between  Ehrlich's  and  Bordet's  views.  Workers  of  the  French 
school  also  hold  that  complement  does  not  exist  in  the  free 
condition  in  the  blood,  but  is  liberated  from  the  leucocytes  when 
the  blood  is  shed.  This  cannot  be  held  as  proved.  On  the 
contrary,  there  are  facts  which  are  strongly  in  support  of  the 
view  that  complement  exists  in  the  free  condition  in  the  circu- 
lating blood.  There  is,  however,  evidence  that  the  amount  of 
free  complement  increases  after  the  blood  is  shed  and  some  time 
later  gradually  diminishes. 

The  hsemolytic  action  of  a  normal  serum  can  be  shown  in  many  cases 
to  be  of  the  same  nature  as  that  of  an  immune-serum,  that  is,  comple- 
ment and  the  homologue  of  an  immune-body  can  be  distinguished.  For 
example,  guinea-pig's  serum  is  hsemolytic  to  ox's  corpuscles  ;  if  a  portion 
of  serum  be  heated  at  55°  C.  the  complement  will  be  destroyed  ;  if 
another  portion  be  treated  with  ox's  corpuscles  at  0°  C.,  the  natural 
immune-body  will  be  removed  and  only  complement  will  be  left. 
Neither  portion  is  in  itself  hremolytic,  but  this  property  becomes  manifest 
again  when  the  two  portions  are  mixed.  Hsemolytic  sera  are  of  great 
service  in  the  study  of  the  question  of  specificity.  Each  is  specific  in  the 
sense  already  explained  (p.  521),  but  the  serum  developed  against  the 
corpuscles  of  an  animal  may  have  some  action  on  those  of  an  allied 
species,  that  is,  some  receptors  are  common  to  the  two  species.  This  fact 
can  be  readily  shown  by  the  usual  absorption  tests,  for  example,  in  the 
case  of  an  anti-ox  serum  tested  on  sheep's  corpuscles.  A  close  analogy 
holds  to  what  has  been  established  in  the  case  of  agglutinins.  It  is 
further  of  great  interest  to  note  that  by  the  injection  of  red  corpuscles 
into  an  animal  its  serum  not  only  becomes  hremolytic,  but  in  many  cases 
when  heated  at  55°  C.  possesses  also  agglutinating  and  opsonic  properties 
towards  the  red  corpuscles  used.  And  further,  it  would  appear  that  in 
some  cases  at  least  the  immune-body,  haemagglutinin,  and  heemopsonin 
are  distinct  substances.  These  facts  abundantly  show  how  close  an 
analogy  obtains  between  anti-bacterial  and  haemolytic  sera,  and  how 
important  a  bearing  hsemolytic  studies  have  on  the  questions  of  im- 
munity in  general. 

In  addition  to   hsemolytic  sera,  anti-sera  have  been  obtained  by  the 


OPSONIC  ACTION  539 

injection  of  leucocytes,  spermatozoa,  ciliated  epithelium,  liver  cells, 
nervous  tissue,  etc.  The  laws  governing  the  production  and  properties 
of  these  are  identical,  that  is,  each  serum  exhibits  a  specific  property 
towards  the  body  used  in  its  production — i.e.  dissolves  leucocytes,  im- 
mobilises spermatozoa,  etc.  The  specificity  is,  however,  not  so  marked 
as  in  the  case  of  sera  produced  against  red  blood  corpuscles  ;  thus 
a  serum  produced  against  tissue  cells  is  often  haemolytic  ;  this  is 
probably  due  to  various  cells  of  the  body  having  the  same  receptors. 
Here  again,  when  the  anti-serum  produces  no  destructive  effect  on  the 
corresponding  cells,  the  presence  of  an  immune-body  may  be  demon- 
strated by  the  increased  amount  of  complement  which  is  taken  up 
through  its  medium.  It  may  also  be  mentioned  that  each  anti-serum 
usually  exhibits  toxic  properties  towards  the  animal  whose  cells  bave 
been  used  in  the  injections,  e.g.  a  hrcmolytic  serum  may  produce  a  fatal 
result,  with  si^ns  of  extensive  blood  destruction,  haemoglobinuria,  etc., 
h.4.  it  is  hiernotoxic  for  the  particular  animal ;  a  serum  prepared  by  in- 
jection of  liver  cells  has  been  found  to  produce  on  injection  necrotic 
changes  in  the  liver  in  the  species  of  animal  whose  liver  cells  were 
used.  These  are  mentioned  as  examples  of  a  very  large  group  of  specific 
activities. 

With  regard  to  the  sites  of  origin  of  immune-bodies  our 
information  is  still  very  deficient.  Pfeiffer  and  Marx  brought 
forward  evidence  in  the  case  of  typhoid,  and  Wassermann  in 
the  case  of  cholera,  that  the  immune-bodies  are  chiefly  formed 
in  the  spleen,  lymphatic  glands,  and  bone-marrow.  According 
to  certain  workers  of  the  French  school,  the  chief  source  of  anti- 
substances  acting  on  cells  such  as  red  blood  corpuscles  is  the 
large  mononucleated  leucocytes,  whilst  those  acting  on  bacteria 
are  chiefly  derived  from  the  polymorpho-nuclear  leucocytes  (vide 
p.  182).  Another  view  is  that  immune-bodies  are  chiefly  formed 
by  the  large  mononucleated  leucocytes,  whilst  complements  are 
products  of  the  polymorphs.  That  these  cells  are  concerned  in  the 
production  of  antagonistic  and  protective  substances  is  almost 
certain,  though  another  possible  source  of  wide  extent,  namely, 
the  endothelium  of  the  vascular  system,  has  been  largely  over- 
looked. 'As  yet,  definite  statements  cannot  be  made  on  thisjpoint. 

(b)  Opsonic  Action. — The  presence  of  a  substance  in  an 
immune-serum  which  makes  the  corresponding  organism  sensi- 
tive to  phagocytosis  was  first  demonstrated  by  Denys  and  Leclef 
in  1895,  in  the  case  of  an  anti-streptococcal  serum.  They  also 
showed  that  the  serum  produced  this  effect  by  acting  on  the 
organism,  not  on  the  leucocytes.  It  is,  however,  chiefly  to  the 
researches  of  Wright  and  his  co-workers  that  this  subject  has 
come  into  special  prominence.  Wright  and  Douglas  -in  their 
first  paper  showed  that  the  phagocytosis  of  staphylococci  by 
leucocytes  depended  on  a  body  in  the  normal  serum  which 
became  fixed  to  the  cocci  and  made  them  a  prey  to  the 


540  IMMUNITY 

phagocytes.  To  this  they  gave  the  name  of  "  opsonin  "  (vide 
pp.  122,  519).  There  is  no  phagocytosis  of  cocci  by  leucocytes 
washed  in  salt  solution ;  normal  serum  heated  to  55°  C.  is  also 
without  effect  in  inducing  this  phenomenon.  They  could  not 
demonstrate  any  effect  of  the  opsonin  on  the  leucocytes.  On  the 
other  hand,  if  bacteria  be  exposed  to  the  fresh  serum,  and  they 
be  freed  from  the  excess  of  serum  and  then  exposed  to  phago- 
cytes also  washed  free  from  serum,  they  will  be  readily  taken 
up  by  the  cells.  It  has  been  abundantly  shown  that  the  opsonic 
action  of  the  serum  is  increased  by  the  process  of  immunisation 
against  an  organism,  and  the  opsonic  index  represents  the 
degree  o'f  immunity  in  one  of  its  aspects  as  already  explained 
(p.  122).  The  matter  has,  however,  become  complicated  by  the 
circumstance  that  in  an  immune-serum  an  opsonin  may  still  be 
present  after  the  serum  is  heated  at  55°  C.,  as  has  been  shown 
by  Dean  and  others ;  and  Muir  and  Martin  have  shown  that 
this  thermostable  immune-opsonin  (bacteriotropin  of  Neufeld) 
has  all  the  specific  characters  of  anti-substances  in  general.  On 
the  other  hand,  they  have  found  that  the  thermolabile  opsonin 
of  a  normal  serum  has  quite  different  properties.  For  example, 
when  a  normal  serum  is  tested  on  a  particular  bacterium,  the 
opsonic  effect  on  that  bacterium  may  be  removed  by  treating  the 
serum  with  other  bacteria;  in  other  words,  the  thermolabile 
opsonin  of  normal  serum  does  not  possess  the  specific  character 
of  the  opsonin  developed  in  the  process  of  immunisation.  They 
have  also  found  that  various  substances  or  combinations  of  sub- 
stances which  act  as  "  complement  absorbers "  also  remove  the 
opsonic  property  from  a  normal  serum,  while  they  have  no  effect 
on  an  immune-opsonin. 

That  this  thermolabile  normal  opsonin  can  act  in  a  non- 
specific way  is  shown  by  the  fact  that  particles  of  car- 
mine and  other  substances  become  opsonised  by  the  action 
of  normal  serum.  It  is,  however,  to  be  noted  that  in  certain 
cases  there  have  been  found  in  a  normal  serum  traces  of  sub- 
stances which  can  be  activated  by  thermolabile  opsonin  after 
the  manner  of  immune-body  and  complement  (as  seen  in  the 
haemolytic  action  of  a  normal  serum  (p.  538) ;  to  this  extent  the 
opsonic  effect  of  a  normal  serum  may  have  some  degree  of 
specificity.  From  this  and  other  facts  some  observers  have 
attempted  to  explain  the  whole  of  opsonic  action  according  to 
the  scheme  of  immune-body  and  complement  as  seen  in  h&mo- 
lysis.  This,  however,  is  not  justifiable, '  since  normal  thermo- 
labile opsonin  can,  as  we  have  seen,  act  by  itself,  as  can  also 
the  specific  immune-opsonin  after  normal  opsonin  has  been 


AGGLUTINATION  541 

destroyed  by  heating,  and  we  know  of  no  corresponding  action  in 
the  case  of  an  immune-body.  The  subject  is  one  of  considerable 
complexity,  but  it  may  be  said  that  the  most  important  varia- 
tions in  the  opsonic  content  observed  in  infections  depend  on 
the  specific  immune-opsonins,  though  the  presence  of  immune- 
body  may  play  a  part  in  raising  the  index  by  leading  to  the 
union  of  more  normal-complement-opsonin. 

Further  study  will  be  necessary  before  the  exact  relationships 
of  these  substances  are  fully  understood,  and  other  questions  with 
regard  to  them  have  jis  yet  scarcely  been  touched  upon. 
Increased  phagocytic  action  had  long  been  known  by  the  work 
.•f  Metelmikotf  to  be  associated  with  the  development  of  active 
immunity,  and  the  theory  of  stimulation  of  leucocytes  was 
supported  liv  many.  The  work  on  opsonins  has  caused  a  swing 
of  the  pendulum  in  the  other  direction,  and  points  to  the 
development  of  anti-substances  in  the  serum  as  the  all-important 
factor.  It  remains  to  be  determined  to  what  extent  the  opsonic 
and  directly  bactericidal  properties  taken  together  will  explain 
the  phenomena  of  natural  and  acquired  immunity. 

(c)  A<i<tliitin<itloi>. — Charrin  and  Roger  in  1889  observed 
that  when  the  bacillus  pyocyaneus  was  grown  in  the  serum  of 
an  animal  immunised  against  this  organism,  the  growth  formed 
a  deposit  at  the  foot  of  the  vessel ;  whereas  a  growth  in  normal 
serum  produced  a  uniform  turbidity.  Griiber  and  Durham,  in 
investigating  Pfeiffer's  reaction,  found  that  when  a  small  quantity 
of  an  anti-serum  is  added  to  an  emulsion  of  the  corresponding 
bacterium,  the  organisms  become  agglutinated  into  clumps,  this 
phenomenon  depending  upon  the  presence  of  definite  bodies  in 
the  serum  called  ayylutinins. 

It  had  been  already  found  that  the  serum  of  convalescents 
from  typhoid  fever  could  protect  animals  to  a  certain  extent 
against  typhoid  fever,  and,  in  view  of  the  facts  experimentally 
established,  it  appeared  a  natural  proceeding  to  inquire  whether 
such  serum  possessed  an  agglutinative  action  and  at  what  stage 
of  the  disease  it  appeared.  The  result,  obtained  independ- 
ently by  Griinbaum  and  Widal,  but  first  published  by  the  latter, 
was  to  show  that  the  serum  possessed  this  specific  action  shortly 
after  infection  had  taken  place ;  in  other  words,  the  develop- 
ment of  this  variety  of  anti-substance  can  be  demonstrated  at 
an  early  stage  of  the  disease.  Agglutination  may  be  said  to  be 
observed  generally  in  bacterial  infections,  though  the  degree  of 
the  phenomenon  and  the  facility  with  which  it  can  be  noted  vary 
greatly  in  different  cases.  Details  will  be  found  in  the  chapters 
dealing  with  the  individual  diseases,  etc.  Furthermore,  the 


542  IMMUNITY 

phenomenon  is  not  peculiar  to  bacteria ;  it  is  seen,  for  example, 
when  an  animal  is  injected  with  the  red  corpuscles  of  another 
species,  hcemagglutinins  appearing  in  the  serum,  which  have  a 
corresponding  specificity. 

The  physical  changes  on  which  agglutination  depends  cannot 
as  yet  be  said  to  be  fully  understood.  Griiber  and  Durham 
considered  that  the  agglutinin  produced  a  change  in  the  envelope 
of  the  bacterium,  causing  it  to  swell  up  and  become  viscous,  but 
the  facts  since  established  show  that  this  is  not  the  true  explana- 
tion. For  example,  it  has  been  shown  by  Nicolle  and  by  Kruse 
that  if  an  old  bacterial  culture  be  filtered  through  porcelain,  the 
addition  of  some  of  the  corresponding  anti-serum  produces  a 
sort  of  granular  precipitate  in  it;  and  that  when,  as  in  the 
agglutination  of  bacteria,  minute  inorganic  particles  are  added 
to  the  mixture,  they  become  aggregated  into  clumps.  The 
phenomenon  would  thus  appear  to  be  the  result  of  the  inter- 
action of  the  agglutinin  and  some  substance  in  the  bacterial  cell 
which  is  known  as  the  agglutinable  substance  or  as  the  agglu- 
tinogen.  Joos  has  found  in  the  case  of  the  typhoid  bacillus  that 
there  are  two  agglutinable  substances,  which  differ  in  their 
resistance  to  heat — a  and  ft  agglutinogen,  and  that  they  give 
rise  to  corresponding  agglutinins.  Further,  as  the  result  of  a 
comparative  study  of  the  agglutinins  of  a  motile  and  a  non- 
motile  variety  of  the  hog  cholera  bacillus,  Theobald  Smith  has 
come  to  the  conclusion  that  there  is  an  agglutinin  which  is  pro- 
duced by  and  acts  on  the  flagella  and  another  which  is  similarly 
related  to  the  bacterial  bodies.  The  former  acts  in  very  much 
higher  dilutions  than  the  latter,  and  this  is  regarded  as  an  ex- 
planation of  the  fact  that  in  the  case  of  non-motile  organisms 
the  agglutinating  serum  acts  only  in  proportionately  high  con- 
centration as  compared  with  the  case  of  most  motile  forms. 
Another  factor  necessary  for  the  phenomenon  of  agglutination 
is  a  proper  salt  content.  Bordet  showed  that  if  the  clumps  of 
agglutinated  bacteria  are  freed  from  salt  by  washing  in  distilled 
water  they  become  resolved,  and  that  on  the  addition  of  some 
sodium  chloride  they  are  formed  again,  and  Joos  has  also  brought 
forward  striking  confirmatory  evidence  as  to  the  necessity  for 
the  presence  of  salts.  It  is  thus  probable  that  in  the  pheno- 
menon of  agglutination  as  ordinarily  understood  more  than  one 
factor  is  concerned,  and  it  is  possible  that  in  part  it  may  depend 
on  some  altered  molecular  relationship  of  the  bacteria  to  the 
surrounding  fluid  analogous  to  altered  surface  tension. 

In  the  phenomenon  of  agglutination  we  have  to  distinguish 
two  factors,  namely,  the  combination  of  agglutinin  and  agglu- 


AGGLUTINATION  543 

tinaKle  sul »st;mce  (agglutinogen)  and  the  actual  clumping  of  the 
bacteria,  and  it  is  to  be  noted  that  whether  or  not  the  latter 
event  follows  depends  on  the  physical  condition  of  each  of  the 
two  substances  concerned.  For  example,  in  some  cases  when 
the  bacteria  are  heated  at  a  temperature  of  65°  C.,  for  some  time, 
they  may  lose  the  faculty  of  being  agglutinated  while  they  may 
still  retain  the  property  of  combining  with  or  binding  agglutinin. 
Dreyer  and  Jex  Blake  have  observed  the  remarkable  fact 
that  in  certain  instances  on  being  heated  to  a  still  higher 
temperature  they  may  once  more  become  agglutinable.  Another 
point  of  practical  importance  is  that  bacteria  when  freshly  grown 
from  the  tissues  are  very  often  less  agglutinable  than  they  after- 
wards become  when  sub-cultured  for  some  time.  As  stated 
above,  the  agglutinins  are  usually  placed  in  the  second  order 
of  anti-substances,  and  are  regarded  as  possessing  a  combining 
group  and  an  active  or  agglutinating  group.  The  constitution 
would  thus  be  analogous  to  that  of  a  toxin,  and  in  conformity 
with  this  view  Eisenberg  and  Volk  consider  that  the  agglutinat- 
ing group  may  be  destroyed  while  the  combining  group 
remains,  the  result  being  an  agglutinoid.  The  evidence  for 
this  lies  in  the  fact  that  when  an  agglutinating  serum  is  heated 
to  a  certain  temperature,  not  only  does  it  lose  its  agglutinating 
action,  but  when  the  bacteria  are  treated  with  such  a  serum 
their  agglutination  by  active  serum  is  interfered  with,  a  sort  of 
plugging  up  of  the  combining  molecules  having  apparently 
taken  place.  Again,  with  agglutinating  sera  partially  inacti- 
vated by  heat  or  other  means,  what  are  known  as  "^ojie_phenQi_ 
mejia "  occur ;  that  is,  when  agglutination  occurs  with  a  given 
dilution  of  such  a  serum  a  lower  dilution  may  fail  to  agglutinate, 
and  this  they  suppose  to  be  due  to  the  interference  of  the  union 
of  agglutinin  by  agglutinoid  in  the  greater  concentration  of 
serum.  On  the  other  hand,  there  are  facts  which  cannot  be 
brought  into  harmony  with  this  view.  For  example,  Dreyer  and 
Jex  Blake  have  shown  that  the  inhibition  zone  may  be  slight 
when  there  has  been  much  destruction  of  agglutinin,  and  on 
the  other  hand  may  be  well  marked  when  no  weakening  of  the 
agglutinating  power  has  resulted  from  the  heating.  The  physical 
changes  underlying  such  phenomena  are  still  very  obscure,  but 
we  may  say  at  present  that  the  existence  of  agglutinoids  has 
not  yet  been  proved. 

Like  immune-bodies,  agglutinins  are  not  destroyed  at  55°  C. 
(a  temperature  sufficient  to  annul  bactericidal  action),  but 
different  agglutinins  show  variations  in  this  respect,  some  being 
affected  by  a  temperature  little  above  that  named.  The  resist- 


544  IMMUNITY 

ance  to  heat  also  varies  when  the  serum  is  diluted  with  salt 
solution,  and  it  has  been  shown  that  conditions  which  interfere 
with  the  coagulation  of  the  proteins  increase  their  resistance. 
Like  antitoxins,  agglutinins  seem  to  be  chiefly  contained  in  the 
globulin  fraction.  Discussion  has  taken  place  as  to  the  relation 
of  agglutinins  to  immune-bodies  and  as  to  how  far  agglutination  is 
an  indication  of  immunity.  It  may  be  said  that  in  the  case  of 
certain  sera  investigated  it  has  been  shown  that  the  immune- 
body  and  the  agglutinin  are  separate  substances,  but  it  would 
not  be  justifiable  to  say  this  is  always  the  case.  And  while  the 
agglutinative  power  cannot  in  itself  be  taken  as  the  measure  of 
the  degree  of  immunity,  agglutinins  and  immune-bodies  are  the 
products  of  corresponding  reactive  processes,  and  their  forma- 
tion is  governed  by  corresponding  laws.  Agglutinins  become 
fixed  in  definite  proportion  by  the  receptors  of  the  bacteria  •  that 
is,  the  agglutinin  becomes  used  up  in  the  process  of  agglutination, 
and  it  has  been  shown  that  bacteria  may  take  up  many  times 
the  amount  necessary  to  their  agglutination — a  corresponding 
fact  to  what  has  been  established  with  regard  to  immune-bodies 
of  hsemolytic  sera.  The  agglutinins  are  specific  in  the  sense 
which  has  been  explained  above  (p.  521).  It  can  be  shown  by 
the  method  of  absorption  that  in  an  agglutinating  serum  there 
may  be  several  agglutinins  with  different  combining  groups,  some 
of  which  may  be  taken  up  by  organisms  allied  to  that  which 
has  given  rise  to  the  anti-serum. 

Besides  those  stated  above,  other  phenomena  have  been 
observed  in  the  interaction  of  anti-sera  and  the  corresponding 
bacteria.  For  example,  it  has  been  shown  that  when  certain 
bacteria — e.g.  the  typhoid  bacillus,  b.  coli,  and  b.  proteus — are 
grown  in  bouillon  containing  a  small  proportion  of  the  homo- 
logous serum,  their  morphological  characters  may  be  altered, 
growth  taking  place  in  the  form  of  threads  or  chains  which  are 
not  observed  in  ordinary  conditions.  In  other  instances  a  serum 
may  inhibit  some  of  the  vital  functions  of  the  corresponding 
bacterium. 

Precipitins. — Shortly  after  the  discovery  of  agglutinins,  Kraus 
showed  in  the  case  of  the  organisms  of  typhoid,  cholera  and 
plague,  that  the  anti-serum  not  only  caused  agglutination,  but 
when  added  to  a  filtrate  of  a  culture  of  the  corresponding 
bacterium  produced  a  cloudiness  and  afterwards  a  precipitate. 
To  the  substance  in  the  immune-serum  which  brought  about 
this  effect  he  gave  the  name  of  precipitin.  Subsequent  study 
has  shown  that  this  phenomenon  is  closely  related  to  agglutina- 
tion ;  in  fact  several  authorities  consider  that  they  represent  the 


SERUM  PRECIPITIN-  545 

same  reaction  under  different  conditions,  that  is,  that  the  sub- 
stances which  when  present  in  the  bacterial  bodies  give  rise  to 
agglutination,  on  the  addition  of  the  anti-serum,  produce  a 
precipitate  when  free  in  a  fluid.  To  test  the  reaction  it  is 
accordingly  necessary  to  have  as  far  as  possible  the  substance  of 
the  bacteria  in  solution,  and  for  this  purpose  there  have  been 
introduced  various  methods,  of  which  the  two  following  may 
be  given : — 

(«)  It  is  well  known  that  in  an  old  bouillon  culture  the  bacteria  undergo 
disintegration  and  their  constituents  go  into  solution.  Accordingly,  if  such 
a  culture  which  has  been  kept  in  the  incubator  for  several  weeks  be 
filtered  through  a  porcelain  filter,  the  filtrate  will  contain  the  interacting 
substance  or  precipitinogen. 

(b)  The  growth  from  a  recent  agar  culture  is  scraped  off  and  suspended 
in  normal  salt  solution,  the  mixture  is  made  feebly  alkaline  with  soda 
solution  and  boiled  for  a  few  minutes.  The  mixture  is  then  neutralised, 
when  a  precipitate  forms,  and  is  filtered  through  filter-paper  ;  the  filtrate 
contains  the  precipitinogen. 

The  test  is  carried  out  by  placing  in  a  number  of  small  test- 
tubes  a  given  amount  of  the  bacterial  nitrate  along  with  varying 
quantities  of  the  homologous  anti-serum.  (The  latter  may  be 
obtained  in  the  usual  way  by  the  repeated  injection  of  dead 
cultures  or  of  bacterial  nitrate.)  As  the  precipitate  forms 
slowly  the  tubes  should  be  placed  in  the  incubator  for  twenty- 
four  hours,  *5  per  cent,  carbolic  acid  being  added  to  prevent  the 
growth  of  bacteria.  This  precipitin  reaction  has  now  been 
observed  in  a  great  many  bacterial  diseases  when  the  patient's 
serum  is  added  to  the  corresponding  bacterial  nitrate,  and  has 
even  been  applied  by  some  observers  as  a  means  of  diagnosis. 
It  is,  however,  less  delicate  and  more  restricted  in  its  applica- 
tion than  the  agglutination  methods. 

Serum  Precipitins.— This  subject  does  not  strictly  belong  to  bacteri- 
ology, but  the  general  phenomena  are  so  closely  allied  to  those  just 
described,  that  some  reference  may  be  made  to  it.  When  the  serum  of 
an  animal  is  injected  in  repeated  doses  into  another  animal  of  different 
species,  after  the  type  of  an  immunisation,  there  appears  in  the  serum  of 
the  animal  treated  a  substance  called  precipitin,  which  causes  a  cloudi- 
ness or  precipitate  when  added  to  the  serum  (precipitinogen)  used.  (In 
the  case  of  rabbits  doses  of  3  to  4  c.c.  of  the  serum  may  be  injected  intra- 
peritoneally  at  intervals  of  four  to  five  days,  a  precipitin  usually  appearing 
at  the  end  of  about  three  weeks.)  The  reaction,  which  is  a  very  delicate 
one,  is  conveniently  observed  by  adding  a  given  amount  of  the  anti- 
serum,  say  '05  c.c.,  to  varying  amounts  of  the  homologous  serum  •!,  '01, 
etc.,  c.c.,  in  a  series  of  small  test  tubes,  the  volume  being  then  made  up 
with  salt  solution  to  1  c.c.  In  this  wa*y  a  definite  reaction  may  be 
observed  with  '001  c.c.  of  the  serum  or  even  less.  Here  again  zone 

35 


546  IMMUNITY 

phenomena,  as  in  the  case  of  agglutination,  are  met  with.  If  the  anti- 
serum  be  heated  to  a  temperature  of  75°  C.  for  some  time  it  acquires 
inhibitory  properties,  so  that  when  added  to  a  mixture  of  serum  and  anti- 
serum  which  would  otherwise  give  a  precipitate,  this  no  longer  occurs. 
Some  observers  consider  that  this  is  due  to  the  presence  of  "  precipitoid  " 
in  the  heated  anti-serum  ;  but  the  observations  of  Welsh  and  Chapman 
show  that  this  view  is  not  in  accordance  with  the  facts,  and  indicate  that 
the  inhibition  is  related  to  a  specific  solvent  action  which  the  heated  anti- 
serum  lias  on  the  precipitate.  They  have  also  shown  that  the  main  mass 
of  the  precipitate  is  furnished  by  the  anti-serum  (precipitin).  and  not  as 
is  usually  supposed  by  the  precipitin  throwing  down  the  protein  of  the 
homologous  serum  ;  this  result  is  of  high  importance  in  connection  with 
the  action  of  anti-substances  in  general.  The  precipitin  reaction  is  specific 
in  the  sense  explained  above.  It  is  always  most  marked  towards  the 
serum  of  the  species  used  in  the  immunisation  ;  but  while  this  is  so, 
there  may  also  be  a  slight  reaction  towards  animals  of  allied  species.  An 
anti-human  serum,  for  example,  gives  the  maximum  reaction  with  human 
serum,  but  also  a  slight  reaction  with  the  serum  of  monkeys,  especially  of 
anthropoid  apes  ;  it,  however,  gives  no  reaction  with  the  serum  of  other 
animals.  The  precipitin  test  has  thus  come  to  be  employed  as  a  means  of 
differentiating  human  from  other  bloods.  Another  interesting  phenomenon 
is  what  is  known  as  the  "  deviation  of  complement,"  which  is  produced  by 
the  combination  of  the  two  substances  in  the  serum  and  anti-serum  respect- 
ively. If  mixtures  be  made  according  to  the  above  method,  and  then  a  small 
quantity  of  complement,  say  fresh  guinea-pig  serum,  be  added,  it  will  be 
found  that  the  complement  becomes  absorbed,  as  may  be  shown  by  sub- 
sequently adding  a  test  amount  of  sensitised  red  blood  corpuscles.  This 
deviation  phenomenon  is  even  a  more  delicate  reaction  than  the  precipitin 
test,  it  being  often  possible  to  demonstrate  by  its  use  from  a  tenth  to  a 
hundredth  of  the  smallest  amount  of  serum  which  will  give  a  perceptible 
precipitate  ;  it  also  is  specific  within  the  same  limits.1 

Therapeutic  Effects  of  Anti-Sera. — As  will  have  been  gathered, 
the  chief  human  diseases  treated  by  anti-sera  are  diphtheria, 
tetanus,  streptococcus  infection,  pneumonia,  dysentery,  plague, 
and  snake  bite.  Of  the  results  of  such  treatment  most  is  known 
in  the  case  of  diphtheria.  Here  a  very  great  diminution  in  the 
mortality  has  resulted.  The  diphtheria  antitoxin  came  into 
general  use  about  October  1894,  and  the  statistics  published  by 
Eehring  towards  the  end  of  1895  indicated  results  which  have 
since  been  confirmed.  In  the  Berlin  Hospitals  the  average 
mortality  for  the  years  1891-93  was  36 '1  per  cent.,  in  1894  it 
was  21 '1  per  cent.,  and  in  January-July  1895,  14'9  per  cent.  The 
objection  that  in  some  epidemics  a  very  mild  type  of  disease 
prevails  is  met  by  the  fact  that  similar  diminutions  of  mortality 
have  occurred  all  over  the  world.  Loddo  collected  the  results 
of  7000  cases  in  Europe,  America,  Australia,  and  Japan,  in 

1  For  an  account  of  precipitins,  vide  Nuttall,  "Blood  Immunity  and 
Relationships,"  Cambridge,  19*04  ;  and  of  complement  deviation,  Muir  and 
Martin,  Journ.  of  Hyg.  (1906),  vi.  p.  265. 


THERAPEUTIC  EFFECTS  OF  ANTI-SERA       547 

which  the  mortality  was  20  per  cent,  as  compared  with  a  former 
mortality  in  the  same  hospitals  of  44  per  cent.  It  has  also  been 
observed  that  if  during  an  epidemic  the  supply  of  serum  fails, 
thr  mortality  at  once  rises;  and  in  two  instances  recorded  it 
was  doubled.  It  must  here  be  ivmt-inbered  that  from  the 
spread  <>f  bacteriological  knowledge  the  diagnosis  of  diphtheria 
is  now  much  more  accurate  than  formerly.  Another  effect  of 
the  antitoxic  treatment  has  been  that  when  tracheotomy  is 
necessary  the  percentage  of  recoveries  is  now  much  higher,  being 
73  per  cent,  instead  of  27  per  cent,  in  a  group  of  cases  collected 
by  the  American  Pediatric  Society.  In  the  London  fever 
hospitals,  since  1894  the  recoveries  after  tracheotomy  have  been 
56 -4  as  compared .  with  32*1  per  cent,  previous  to  the  intro- 
duction of  antitoxin.  One  of  the  most  striking  results  obtained 
in  the  same  hospitals  is  a  reduction  of  the  death-rate  in  post- 
scarlatinal  diphtheria  from  50  per  cent,  to  between  4  per  cent, 
and  5  per  cent.  As  the  disease  here  occurs  while  the  patient  is 
under  observation,  the  treatment  is  nearly  always  begun  on  the 
first  day.  It  is  a  matter  of  prime  importance  that  the  treat- 
ment should  be  commenced  whenever  the  disease  is  recognised. 
1  >fh ring  showed  that  in  cases  treated  on  the  first  and  second 
days  of  the  disease  the  mortality  was  only  7 '3  per  cent.,  and  this 
has  been  generally  confirmed,  whilst  after  the  fifth  day  it  was  of 
little  service  to  apply  the  treatment.  In  order  to  obtain  such 
results,  it  cannot  be  too  strongly  insisted  on  that  attention 
should  be  given  to  the  dosage.  When  bad  results  are  obtained, 
it  may  be  strongly  suspected  that  this  precaution  has  not  been 
observed.  In  the  treatment  of  acute  tetanus  by  the  antitoxin 
the  improvement  in  results  has  not  been  marked,  but  some 
chronic  cases  have  been  benefited,  and,  as  already  stated  (p.  431) 
better  results  are  obtained  in  acute  cases  if  intravenous  in- 
jection be  practised.  In  the  case  of  Yersin's  anti-plague  serum, 
though  some  benefit  has  appeared  to  follow  its  use,  this  has 
been  of  quite  a  limited  nature.  The  same  may  be  said  to  be 
true  of  the  anti-streptococcic  and  anti-pneumonic  sera,  though 
in  the  case  of  the  first  mentioned  numerous  cases  of  apparently 
successful  result  have  been  recorded.  With  regard  to  anti- 
venin,  Lamb  has  shown  that,  if  a  cobra  with  full  glands  bites  a 
man,  many  times  the  minimal  lethal  dose  are  probably  injected. 
In  cases  of  slight  bite,  however,  benefit  may  accrue  from  the  use 
of  the  anti-serum. 

As  has  been  shown  above,  antibacterial  sera  require  for  their 
bactericidal  action  a  sufficiency  of  complement,  and  as  this 
diminishes  in  amount  when  a  serum  is  kept,  the  unsatisfactory 


548  IMMUNITY 

results  with  this  class  of  sera  may  be  due  to  a  deficiency  of 
complement.  Or  it  may  be  as  Ehrlich  has  suggested,  that  the 
complement  naturally  existing  in  human  serum  does  not  suit 
the  immune-body  in  the  anti-serum — that  is,  is  not  taken  up 
through  the  medium  of  the  latter  and  brought  into  combination 
with  the  bacterium.  And  there  is  still  the  further  possibility 
that  even  though  the  complement  should  be  taken  up,  the 
zymotoxic  group  of  the  latter  is  not  sufficiently  active  towards 
the  bacterium  to  effect  its  death.  In  both  cases  it  will  appear 
that  an  extracellular  bactericidal  action  cannot  be  produced  by 
the  particular  immune-body  in  association  with  the  complement 
of  the  animal  in  question.  There  is  no  doubt  that  this  question 
of  complements  is  one  of  high  importance,  and  that  both  com- 
bining affinity  and  toxic  action  of  complements  must  be  con- 
sidered in  each  case. 

Theories  as  to  Acquired  Immunity. 

The  advances  made  within  recent  years  in  our  knowledge 
regarding  artificial  immunity,  and  the  methods  by  which  it  may 
be  produced,  have  demonstrated  the  insufficiency  of  various 
theories  which  had  been  propounded.  Only  a  short  reference 
need  be  made  to  these.  The  theory  of  exhaustion,  with  which 
Pasteur's  name  is  associated,  supposed  that  in  the  body  of  the 
living  animal  there  are  substances  necessary  for  the  existence  of 
a  particular  organism,  which  become  used  up  during  the  sojourn 
of  that  organism  in  the  tissues ;  this  pabulum  being  exhausted, 
the  organisms  die  out.  Such  a  supposition  is,  of  course,  quite 
disproved  by  the  facts  of  passive  immunity.  According  to  the 
theory  of  retention,  the  bacteria  within  the  body  were  considered 
to  produce  substances  which  are  inimical  to  their  growth,  so  that 
they  die  out,  just  as  they  do  in  a  test-tube  culture  before  the 
medium  is  really  exhausted.  Such  a  theory  only  survives  now 
in  the  view  that  antitoxins  are  modified  toxins,  the  evidence 
against  which  has  already  been  discussed  (p.  530).  There  then 
came  the  humoral  theory  and  the.  theory  of  phagocytosis,  but 
neither  of  these  is  tenable  in  its  pure  form,  and  the  distinction 
between  them  need  not  be  maintained.  For,  on  the  one  hand, 
any  substance  with  specific  property  in  the  serum  must  be  the 
product  of  cellular  activity,  and,  on  the  other  hand,  the  facts 
with  regard  to  passive  immunity  go  far  beyond  the  ingestive  and 
digestive  properties  of  phagocytes,  though  these  cells  may  be  in 
part  the  source  of  important  bodies  in  the  serum.  At  the  pre- 
sent time  interest  centres  around  two  theories,  namely,  Ehrlich 's 


EHRLICH'S  SIDE-CHAIN  THEORY  549 

side-chain  theory  and  Metchnikoff s  phagocytic  theory  as  further 
developed.  These  will  now  be  discussed,  and  it  may  be  noted 
that  the  ground  covered  by  each  is  not  coextensive.  For  the 
former  deals  chiefly  with  the  production  of  anti-substances  and 
its  biological  significance,  the  latter  deals  with  the  defensive 
pro  forties  of  cells,  either  directly  by  their  phagocytic  activity 
or  indirectly  by  substances  produced  by  them  after  the  manner 
of  digestive  ferments.  It  will  be  seen,  however,  that  each  has 
a  normal  process  as  its  basis,  namely,  that  of  nutrition. 

1.  Ehrlich's  Side-Chain  Theory. — This  may  be  said  to  be  an 
application  of  his  views  regarding  the  nourishment  of  proto- 
plasm. A  molecule  of  protoplasm  (in  the  general  sense)  may  be 
regarded  as  composed  of  a  central  atom  group  or  executive 
centre  (Leistungskern)  with  a  large  number  of  side-chains 
(Seitenketten),  i.e.  atom  groups  with  combining  affinity  for 
food-stuffs.  It  is  by  means  of  these  latter  that  the  living 
molecule  is  increased  in  the  process  of  nutrition,  and  hence 
the  name  receptors  given  by  Ehrlich  is  on  the  whole  preferable. 
These  receptors  are  of  three  chief  kinds  corresponding  to  the 
classes  of  anti-substances  described  (p.  521);  the  first  has  a 
single  unsatisfied  combining  group,  and  merely  fixes  molecules 
of  relatively  simple  constitution — receptor  of  the  first  order ; 
the  second  has  a  combining  group  for  the  food  molecule,  and 
another  active  or  zymotoxic  group,  which  leads  to  some  physical 
change  in  it — receptor  of  the  second  order ;  the  third  has  two 
combining  groups,  one  for  the  food  molecule  and  another  which 
fixes  a  ferment '(or  complement)  in  the  fluid  medium  around — 
receptor  of  the  third  order  or  amboceptor.  These  latter  receptors 
come  into  action  in  the  case  of  larger  food  molecules  which 
require  to  be  broken  up  by  ferment-action  for  the  purposes 
of  the  cell  economy.  In  considering  the  application  of  this  idea 
to  the  facts  of  acquired  immunity,  it  must  be  kept  in  view  that 
all  the  substances  to  which  anti-substances  have  been  obtained 
are,  like  proteids,  of  unknown  but  undoubtedly  of  very  complex 
chemical  constitution,  and  that  in  apparently  every  case  the 
;mti -substance  enters  into  combination  with  its  corresponding 
substance  antigen.  The  dual  constitution  of  toxins  and  kindred 
substances,  as  already  described  (p.  198),  is  also  of  importance  in 
this  connection.  Now,  to  take  the  case  of  toxins,  when  these 
aiv  introduced  into  the  system  they  are  fixed,  like  fund  stiitt's, 
1>\  tlirir  h;i|>ti>]iliorous  groups  to  the  receptors  of  the  cell 
protoplasm,  but  are  unsuitable  for  assimilation.  If  they  are  in 
sutliciently  large  amount,  the  toxophorous  part  of  the  toxin 
molecule  produces  that  disturbance  of  the  protoplasm  which 


550  IMMUNITY 

is  shown  by  symptoms  of  poisoning.  If,  however,  they  are 
in  smaller  dose,  as  in  the  early  stages  of  immunisation,  fixation 
to  the  protoplasm  occurs  in  the  same  way;  and  as  the  com- 
bination of  receptors  with  toxin  is  supposed  to  be  of  firm 
nature,  the  receptors  are  lost  for  the  purposes  of  the  cell,  and 
the  combination  K.-T.  (receptor  +  toxin)  is  shed  off  into  the 
blood.  The  receptors  thus  lost  become  replaced  by  new  ones, 
and  when  additional  toxin  molecules  are  introduced,  these  new 
receptors  are  used  up  in  the  same  manner  as  before.  As  a  result  of 
this  repeated  loss  the  regeneration  of  the  receptors  becomes  an  over- 
regeneration,  and  the  receptors  formed  in  excess  appear  in  the 
free  condition  in  the  blood  stream  and  then  constitute  antitoxin 
molecules.  There  are  thus  three  factors  in  the  process,  namely, 
(1)  fixation  of  toxin,  (2)  over-production  of  receptors,  (3)  set- 
ting free  of  receptors  produced  in  excess.  Accordingly  these  re- 
ceptors which,  when  forming  part  of  the  cell  protoplasm,  anchor 
the  toxin  to  the  cell,  and  thus  are  essential  to  the  occurrence  of 
toxic  phenomena,  in  the  free  condition  unite  with  the  toxin,  and 
thus  prevent  the  toxin  from  combining  with  the  cells  and  exert- 
ing a  pathogenic  action.  The  three  orders  of  receptors,  when 
separated  from  the  cells,  thus  give  the  three  kinds  of  anti- 
substances.  Ehrlich  does  not  state  what  cells  are  specially 
concerned  in  the  production  of  anti-substances,  but  from  what 
has  been  stated  it  is  manifest  that  any  cell  which  fixes  a  toxin 
molecule,  for  example*,  is  potentially  a  source  of  antitoxin. 
Cells,  to  whose  disturbance,  resulting  from  the  fixation  of  toxin, 
characteristic  symptoms  of  poisoning  are  due,  will  thus  be 
sources  of  antitoxin,  e.g.  cells  of  the  nervous  system  in  the  case 
of  tetanus,  though  the  cells  not  so  seriously  affected  by  toxin 
fixation  may  act  in  the  same  way.  The  experimental  investiga- 
tion of  the  source  of  antitoxins  has,  however,  yielded  little  result, 
and  no  definite  statement  can  be  made  on  the  subject. 

When  we  come  to  consider  how  far  Ehrlich's  theory  is  in 
harmony  with  known  facts,  we  find  that  there  is  much  in  its 
favour.  In  the  first  place,  it  explains  the  difference  between 
active  and  passive  immunity,  e.g.  difference  in  duration,  etc. ;  in 
the  former  the  cells  have  acquired  the  habit  of  discharging  anti- 
substances,  in  the  latter  the  anti-substances  are  simply  present 
as  the  result  of  direct  transference.  It  is  also  in  harmony  with 
the  action  of  antitoxins,  etc.,  as  detailed  above,  and  especially 
it  affords  an  explanation  of  the  multiplicity  of  anti-substances. 
For,  if  we  take  the  case  of  antitoxins,  we  see  that  this  depends 
upon  the  combining  affinity  of  the  toxin  for  certain  of  the  cells 
of  the  body,  and  this  again  is  referred  back  to  the  complicated 


EHRLICH'S  SIDE-CHAIN  THEORY  551 

constitution  of  living  protoplasm.  Furthermore,  the^  biological 
principle  involved  is  no  new  one,  being  simply  that  of  over- 
regeneration  after  loss.  It  would  appear  likely  that  the  integrity 
of  the  executive  centres  of  the  protoplasm  molecules  would  be 
essential  to  the  satisfactory  production  of  side-chains,  and  this 
would  appear  in  accordance  with  the  fact  that  antitoxin 
formation  occurs  most  satisfactorily  when  there  is  no  marked 
disturbance  of  the  health  of  the  animal. 

It  is  to  be  noted,  however,  that  it  does  not  explain  active 
immunity  apart  from  the  presence  of  anti-substances  in  the 
serum.  For  example,  an  animal  may  be  able  to  withstand  a 
much  larger  amount  of  toxin  than  could  be  neutralised  by  the 
total  amount  of  antitoxin  in  its  serum.  This  might  theoretically 
be  explained  by  supposing  a  special  looseness  of  the  cell  re- 
ceptors so  that  the  toxin-receptor  combination  became  readily  cast 
oft'.  The  question,  however,  arises  whether  there  may  not  be 
really  an  increased  resistance  of  the  cells  to  the  toxophorous 
affinities.  An  observation  made  by  Meyer  and  Ransom  (vide 
p.  427)  is  also  difficult  of  explanation,  according  to  the  view 
that  antitoxin  is  formed  by  the  cells  with  which  the  toxin 
combines  and  on  which  it  acts.  They  found  that  in  an  animal 
actively  immunised  against  tetanus  and  with  antitoxin  beginning 
to  appear  in  its  blood,  the  injection  of  a  single  M.L.D.  of 
tetanus  toxin  into  a  peripheral  nerve  brought  about  tetanus 
with  a  fatal  result.  On  the  other  hand,  the  injection  of  anti- 
toxin into  the  sciatic  nerve  above  the  point  of  injection  of  toxin 
prevented  the  latter  from  reaching  the  cells  of  the  cord.  One 
can  scarcely  imagine  an  explanation  of  these  facts  if  antitoxin 
molecules  were  in  process  of  being  shed  off  by  the  cells  of  the 
nervous  system.  Further,  when  the  serum  of  an  animal  con- 
tains a  large  amount  of  antitoxin,  how  does  the  additional 
toxin  injected  reach  the  cells  in  order  to  influence  them  as 
we  know  it  does'?  This  also  is  difficult  to  understand,  unless 
the  toxin  has  a  greater  affinity  for  the  receptors  in  the  cells 
than  for  the  free  receptors  (antitoxin)  in  the  serum.  A  super- 
sensitiveness  of  the  nerve-cells  of  an  animal  to  tetanus  toxin, 
sometimes  observed  even  when  there  is  a  large  amount  of 
antitoxin  in  the  serum,  has  been  often  brought  forward  as  an 
objection.  But  this  also  may  perhaps  be  explained  by  there 
having  occurred  a  partial  damage  of  the  cell  protoplasm 
by  the  toxic  action  in  the  process  of  immunisation  —  an 
explanation  which,  of  course,  demands  that  in  some  way  the 
fiv-hly  introduced  toxin  may  reach  the  cells  in  spite  of  the  anti- 
toxin in  the  blood,  or  it  may  belong  to  the  group  of  anaphy lactic 


552  IMMUNITY 

phenomena  described  below  (p.  558).  Further  investigation  alone 
will  settle  these  and  various  other  disputed  points,  and  may 
remove  many  of  the  apparent  objections.  At  present  we  may 
say,  however,  that  Ehrlich's  theory  is  the  only  one  which  even 
attempts  to  explain  the  cardinal  facts  of  this  aspect  of  immunity. 
2.  The  Theory  of  Phagocytosis. — This  theory,  brought 
forward  by  Metchnikoff  to  explain  the  facts  of  natural  and 
acquired  immunity,  has  been  of  enormous  influence  in  stimu- 
lating research  on  the  subject.  Looking  at  the  subject  from  the 
standpoint  of  the  comparative  anatomist,  he  saw  that  it  was 
a  very  general  property  possessed  by  certain  cells  throughout 
the  animal  kingdom,  that  they  should  take  up  foreign  bodies 
into  their  interior  and  in  many  cases  digest  and  destroy  them. 
On  extending  his  observations  to  what  occurred  in  disease,  he 
came  to  the  conclusion  that  the  successful  resistance  of  an 
animal  against  bacteria  depended  on  the  activity  of  certain  cells 
called  phagocytes.  In  the  human  subject  he  distinguished  two 
chief  varieties,  namely — (a)  the  microphages,  which  are  the 
"  polymorpho-nuclear  "  finely  granular  leucocytes  of  the  blood ; 
and  (b)  the  macrophages,  which  include  the  larger  hyaline 
leucocytes,  endothelial  cells,  connective  tissue  corpuscles,  and,  in 
short,  any  of  the  larger  cells  which  have  the  power  of  ingesting 
bacteria.  Insusceptibility  to  a  given  disease  is.  indicated  by  a 
rapid  activity  on  the  part  of  the  phagocytes,  different  varieties 
being  concerned  in  different  cases, — an  activity  which  may 
rapidly  destroy  the  bacteria  and  prevent  even  local  damage.  If 
the  organisms  are  introduced  into  the  tissues  of  a  moderately 
susceptible  animal,  there  occurs  an  inflammatory  reaction  with 
local  leucocytosis,  which  results  in  the  intracellular  destruction 
of  the  invading  organisms.  Phagocytosis  is  regarded  by 
Metchnikoff  as  the  essence  of  inflammation.  He  also  showed 
that  the  bacteria  may  be  in  a  living  and  active  state  when  they 
are  ingested  by  leucocytes.  On  the  other  hand,  he  found  that 
in  a  susceptible  animal  phagocytosis  did  not  occur  or  was  only 
imperfect.  He  also  showed  that  when  a  naturally  susceptible 
animal  was  immunised,  the  process  was  accompanied  by  the 
appearance  of  an  active  phagocytosis.  The  ingestion  of  bacteria 
by  phagocytes  is  undoubtedly  a  phenomenon  of  the  greatest 
importance  in  the  defence  of  the  organism.  It  is  known  that 
amoebae  and  allied  organisms  have  digestive  properties  which 
are  specially  active  towards  bacteria,  and  from  what  can  be 
directly  observed,  as  well  as  indirectly  inferred,  there  can  be  no 
doubt  that  such  a  faculty  is  also  possessed  by  the  phagocytes  of 
the  body.  Thus  bacteria  within  these  cells  are  in  a  position 


THE  THEORY  OF  PHAGOCYTOSIS     553 

favourable  to  their  destruction,  and  do  in  many  instances 
become  destroyed.  In  fact,  observations  on  phagocytosis  in 
vitro  show  that  such  destruction  may  in  the  case  of  some 
organisms  occur  so  rapidly  that  the  actual  number  observable  in 
the  leucocytes  is  no  indication  of  the  activity  of  the  process. 
In  other  instances,  e.g.  in  gonorrhcea,  the  ingested  organisms 
would  appear  to  survive  a-  considerable  time  without  undergoing 
change.  Undoubtedly  phagocytosis  is  of  the  highest  importance 
in  active  immunity,  as  by  its  means  organisms  which  would  not 
undergo  an  extra-cellular  death  may  be  killed  off.  In  the  process 
of  immunisation  of  a  susceptible  animal  we  see  a  negative  or 
neutral  chemiotaxis  becoming  replaced  by  positive  chemiotaxis. 
This  has  been  explained  by  Metchnikoff  as  due  to  an  education 
or  stimulation,  of  the  phagocytes.  The  recent  work  on  opsonins 
shows,  however,  that  this  is  not  the  case,  as  leucocytes  from  an 
immunised  animal  are  not  more  active  in  this  direction  than 
those  of  a  normal  animal,  the  all-important  factor  being  the 
development  of  an  opsonin  in  the  immune  animal.  Thus  this 
phase  of  immunity  comes  to  be  merely  a  part  of  the  subject  of 
anti-substances  in  general. 

The  digestive  ferments  of  phagocytes  or  cytases  are,  according 
to  Metchnikoff,  retained  within  the  cells  under  normal  conditions, 
but  are  set  free  when  these  cells  are  injured,  for  example,  when 
the  blood  is  shed.  They  then  become  free  in  the  serum  by 
the  breaking  up  of  the  cells — the  process  known  as  phagolysis — 
and  they  then  constitute  the  alexines,  or  complements  of  Ehrlich. 
Of  these,  as  has  already  been  said,  Metchnikoff  thinks  there 
are  probably  two  kinds — one  called  macrocytase,  contained  in 
the  macrophages,  which  is  specially  active  toward  the  formed 
elements  of  the  animal  body,  protozoa,  etc. ;  and  the  other, 
microcytase,  contained  within  the  polymorpho-nuclear  leucocytes, 
which  has  a  special  digestive  action  on  bacteria.  It  is  the 
microcytase  which  gives  blood  serum  its  bactericidal  properties. 
It  api>ears  to  us,  however,  that  Metchnikoff  has  gone  too  far  in 
distinguishing  the  activities  of  the  two  classes  of  cells  so  much 
as  he  has  done. 

When  the  properties  of  antibacterial  sera,  as  above  described, 
are  considered  in  relation  to  phagocytosis,  Ifetehnikoff  irives  the 
following  explanation.  He  admits  that  the  Lmnrane-body  is 
fixed  by  the  bacteria  (or  red  corpu><-lr-.  aa  the  ease  may  be), 
though  he  doe-  n«.t  state  that  a  chemical  combination  takes 
place ;  hence  In-  calls  it  a  fixative  (Jisateur).  The  immune-bodied 
are  to  be  regarded  as  auxiliary  ferments  (ferments  adjuvants) 
which  aid  the  action  of  the  alexine.  Unlike  the  latter,  however, 


554  IMMUNITY 

they  are  formed  in  excess  during  immunisation  and  set  free  in 
the  serum.  He  compares  their  action  to  that  of  enterokinase, 
a  ferment  which  is  produced  in  the  intestine  and  which  aids 
the  action  of  trypsin.  Thus,  when  the  bacteria  have  fixed  the 
immune-body,  their  digestion  is  facilitated  either  within  the 
phagocytes,  or  outside  of  them  when  the  alexine  has  been  set 
free  by  phagolysis.  He,  however,  maintains  that  extracellular 
digestion  or  lysogenesis  does  not  take  place  without  the 
occurrence  of  phagolysis.  The  source  of  immune-bodies  is,  in 
all  probability,  also  the  leucocytes,  as  these  substances  are 
specially  abundant  in  organs  rich  in  such  cells — spleen,  lymphatic 
glands,  etc. ;  here  again  the  mono-nuclear  leucocytes  are  probably 
the  source  of  the  immune-bodies  concerned  in  haemolysis,  the 
polymorpho-nuclear  leucocytes  the  source  of  those  concerned 
in  bacteriolysis.  Although  the  immune-bodies  are  usually 
set  free  in  the  serum,  this  is  not  always  the  case ;  sometimes 
they  are  contained  in  the  cells,  and  this  probably  occurs  when 
there  is  a  high  degree  of  active  immunity  against  bacteria 
without  a  serum  having  an  antibacterial  action,  the  powers  of 
intracellular  digestion  being  in  such  cases  increased.  In  this 
way  the  facts  of  immunity  can  be  explained  so  far  as  these 
concern  the  destruction  of  bacteria. 

MetchnikofFs  work  has  less  direct  bearing  on  the  production  of 
antitoxins.  He  admits  the  fixation  of  the  toxin  by  the  antitoxin 
to  form  a  neutral  compound,  and  he  apparently  considers  that 
leucocytes  may  also  be  concerned  in  the  production  of  antitoxins. 
Apart,  however,  from  antitoxin  formation,  he  considers  the 
acquired  resistance  of  the  cells  themselves  of  high  importance 
in  toxin  immunity. 

When  we  consider  Metchnikoff's  theory  as  thus  extended  to 
cover  recently  established  facts,  it  must  be  admitted  that  it 
affords  a  rational  explanation  of  a  considerable  part  of  the 
subject,  though  the  elucidation  of  the  chemiotactic  phenomena 
during  immunisation  as  explained  above  detracts  from  the  im- 
portance which  he  attached  to  the  leucocyte.  It,  however,  does 
not  afford  explanation  of  the  multiplicity  and  specificity  of 
antitoxins  as  Ehrlich's  does;  on  the  other  hand,  it  is  more 
concerned  with  the  cells  of  the  body  as  destroyers  or  digesters  of 
bacteria.  As  regards  the  subject  of  antibacterial  sera,  the  results 
of  these  two  workers  may  be  said  to  be  in  harmony  in  some  of 
the  fundamental  conceptions.  And  it  is  of  interest  to  note 
'that  Metchnikoff,  starting  with  the  phenomena  of  intracellular 
digestion,  has  arrived  at  the  giving  off  of  specific  ferments  by 
phagocytes ;  whilst  Ehrlich,  from  his  first  investigations  on  the 


NATURAL  BACTERICIDAL  POWERS  555 

constitution  of  toxins,  has  arrived  at  an  explanation  of  antitoxins 
and  immune-bodies  also  with  a  theory  of  cell-nutrition  as  its 
basis.  Within  the  last  few  years  marked  progress  has  thus 
been  made  towards  the  establishment  of  the  fundamental  laws 
of  immunity. 

NATURAL  IMMUNITY. 

\\Y  have  placed  the  consideration  of  this  subject  after  that  of 
acquired  immunity,  as  the  latter  supplies  facts  which  indicate  in 
what  direction  an  explanation  of  the  former  may  be  looked  for. 
There  may  be  said  to  be  two  main  facts  with  regard  .to  natural 
immunity.  The  first  is,  that  there  is  a  large  number^of  bacteria 
— the  so-called  non-pathogenic  organisms — which  are  practically 
incapable,  unless  perhaps  in  very  large  doses,  of  producing  patho- 
genic effects  in  any  animal ;  when  these  are  introduced  into  the 
body  they  rapidly  die  out.  This  fact,  accordingly,  shows  that 
the  animal  tissues  generally  have  a  remarkable  power  of  destroy- 
ing living  bacteria.  The  second  fact  is,  that  there  are  other 
bacteria  which  are  very  virulent  to  some  species  of  animals, 
whilst  they  are  almost  harmless  to  other  species;  the  anthrax 
bacillus  may  be  taken  as  an  example.  Now  it  is  manifest  that 
natural  immunity  against  such  an  organism  might  be  due  to  a 
special  power  possessed  by  an  animal  of  destroying  the  organisms 
when  introduced  into  its  tissues.  It  might  also  possibly  be  due 
to  an  insusceptibility  to,  or  power  of  neutralising,  the  toxins  of 
the  organism.  For  the  study  of  the  various  diseases  shows  that 
the  toxins  (in  the  widest  sense)  are  the  weapons  by  which  morbid 
changes  are  produced,  and  that  toxin-formation  is  a  property 
common  to  all  pathogenic  bacteria.  There  is,  moreover,  no 
such  thing  known  as  a  bacterium  multiplying  in  the  living  tissues 
without  producing  local  or  general  changes,  though,  theoretically, 
there  might  be.  As  a  matter  of  fact,  however,  natural  immunity 
is  in  most  cases  one  against  infection,  i.e.  consists  in  a  power 
possessed  by  the  animal  body  of  destroying  the  living  bacteria 
\\  In -n  introduced  into  its  tissues  :  such  a  power  may  exist  though 
the  animal  is  still  susceptible  to  the  separated  toxins.  We  shall 
now  look  at  these  two  factors  separately. 

1.  Variations  in  Natural  Bactericidal  Powers. — The  funda- 
mental fact  here  is  that  a  given  bacterium  may  be  rapidly 
destroyed  in  one  animal,  whereas  in  another  it  may  rapidly 
multiply  and  produce  morbid  effects.  The  special  powers  of 
destroying  organisms  in  natural  immunity  have  been  ascribed 
to  (a)  phagocytosis,  and  (b)  the  action  of  the  serum. 

(a)  The  chief  factors  with  regard  to  phagocytosis  have  been 


556  IMMUNITY 

given  above.  The  bacteria  in  a  naturally  immune  animal,  for 
example,  the  anthrax  bacillus  in  the  tissues  of  the  white  rat,  are 
undoubtedly  taken  up  in  large  numbers  and  destroyed  by  the 
phagocytes,  whereas  in  a  susceptible  animal  this  only  occurs  to 
a  small  extent ;  and  Metchnikoff  has  shown  that  they  are  taken 
up  in  a  living  condition,  and  are  still  virulent  when  tested  in  a 
susceptible  animal.  Variations  in  phagocytic  activity  are  found 
to  correspond  more  or  less  closely  with  the  degree  of  immunity 
present,  but  are  probably  in  themselves  -capable  of  explanation. 
The  fundamental  observations  of  Wright  and  Douglas  show  that 
in  many  cases  at  least,  leucocytes  •  do  not  ingest  organisms  in 
normal  saline  solution,  and  that  this  is  not  due  to  the  medium 
in  which  they  are,  is  readily  shown  by  subjecting  the  organisms 
to  the  action  of  fresh  serum  and  then  washing  them ;  thereafter, 
they  are  rapidly  taken  up  by  the  leucocytes  in  salt  solution. 
In  most  cases  this  result  is  due  to  the  labile  opsonin  of  normal 
serum  which  has  combining  affinities  for  a  great  many  organisms, 
as  already  stated.  In  other  cases  more  specific  substances  may 
be  concerned.  But  the  all-important  fact  is  that  whether 
phagocytosis  occurs  or  not,  appears  to  depend  upon  certain  bodies 
in  the  serum.  As  yet  we  cannot  say  whether  the  phagocytosis 
in  a  given  serum,  observed  according  to  the  opsonic  technique, 
always  runs  parallel  with  phagocytosis  in  the  tissues  of  the 
animal  from  which  the  serum  has  been  taken.  This  is  a  subject 
on  which  extended  observations  are  necessary.  But  whether  or 
not  phagocytosis  in  vivo  corresponds  with  that  in  vitro  it  is 
probably  to  be  explained  in  the  same  Avay ;  that  is,  it  probably 
depends  upon  the  content  of  the  serum.  The  composition  of  the 
latter,  no  doubt,  is  the  result  of  cellular  activity,  and  in  this 
the  leucocytes  themselves  are  in  all  probability  concerned,  but 
the  movements  and  phagocytic  activity  of  these  cells  seem  to 
be  chiefly  if  not  entirely  controlled  by  their  environments. 
Ingestion  is,  however,  only  the  first  stage  in  the  process ;  intra- 
cellular  destruction  is  the  second,  and  is  of  equal  importance. 
What  may  be  called  intracellular  bactericidal  action  probably 
varies  in  the  case  of  leucocytes  of  different  animals,  but  regarding 
this  our  knowledge  is  deficient,  and,  further,  bacteria  may  some- 
times survive  the  cells  which  have  ingested  them. 

(6)  When  it  had  been  shown  that  normal  serum  possessed 
bactericidal  powers  against  different  organisms,  the  question 
naturally  arose  as  to  whether  this  bactericidal  power  varied  in 
different  animals  in  proportion  to  the  natural  immunity  enjoyed 
by  them.  The  earlier  experiments  of  Behring  appeared  to  give 
grounds  for  the  belief  that  this  was  the  case.  He  found,  for 


NATURAL  SUSCEPTIBILITY  TO  TOXINS       557 

example,  that  the  serum  of  the  white  rat,  which  has  a  remark- 
able immunity  to  anthrax,  had  greater  bactericidal  powers  than 
that  of  other  animals  investigated.  Further  investigation,  how- 
ever, has  shown  that  this  is  not  an  example  of  a  general  law, 
and  that  the  bactericidal  action  of  the  serum  does  not  vary  pari 
jrttMn  with  the  degree  of  immunity.  In  many  cases,  however, 
non-pathogenic  and  also  attenuated  pathogenic  bacteria  can  be 
M  VM  to  undergo  rapid  solution  and  disappear  when  placed  in  a 
drop  of  normal  serum.  The  bactericidal  action  of  the  serum 
wus  specially  studied  by  Nuttall,  and  later  by  Buchner  and 
Hankin,  who  believe  that  the  serum  owes  its  power  to  certain 
substances  in  it  derived  from  the  spleen,  lymphatic  glands, 
thymus,  and  other  tissues  rich  in  leucocytes.  To  these 
substances  Buchner  gave  the  name  of  alexines ;  as  already 
explained,  they  correspond  with  Metchnikoffs  cytases  and 
Ehrlich's  complements  described  above.  They  can  be  pre- 
cipitated by  alcohol  and  by  ammonium  sulphate,  and  in  this 
respect  and  in  their  relative  lability  correspond  with  enzymes  or 
unorganised  ferments.  Variations  in  bactericidal  power  of  the 
serum  as  tested  in  vitro,  however,  do  not  explain  the  presence 
or  absence  of  natural  immunity  against  a  living  bacterium.  In 
some  cases,  for  example,  it  has  been  found  to  be  considerable, 
while  the  organisms  nourish  in  the  body,  and  the  animal  has  no 
immunity.  In  such  a  case  Metchnikoff  says  that  there  occurs  in 
the  living  body  no  liberation  of  alexines  by  the  phagocytes,  and 
hence  no  bactericidal  action  such  as  occurs  when  the  blood  is 
shed.  In  the  case  of  the  hsemolytic  action  of  a  normal  serum, 
it  has  been  shown  in  many  instances  that  in  addition  to  com- 
plement a  natural  immune-body  is  also  concerned  (p.  534),  and 
this  would  appear  to  be  the  rule  ;  the  process  being  analogous  to 
what  is  seen  in  the  case  of  an  artificially  developed  haemolytic 
serum.  In  certain  instances  an  analogous  condition  appears  to 
obtain  in  a  normal  bactericidal  serum.  For  example  the  dog's 
serum  heated  at  58°  C.  contains  a  natural  immune-body  to 
anthrax  which  can  be  activated  by  the  addition  of  normal 
guinea-pig's  serum  so  as  to  produce  a  bactericidal  action,  though 
the  latter  is  by  itself  without  any  such  effect.  At  present,  how- 
ever, the  possibility  of  bactericidal  action  by  complement  alone 
cannot  be  excluded,  as  it  appears  to  combine  with  many  bacteria 
without  any  intermediary.  Further  work  is  necessary  to  deter- 
mine whether  all  the  facts  regarding  natural  immunity  are  ex- 
plainable by  the  opsonic  and  bactericidal  properties  of  the  serum. 
2.  Variations  in  Natural  Susceptibility  to  Toxins. — We  must 
here  start  with  the  fundamental  fact,  incapable  of  explanation, 


558  IMMUNITY 

that  toxicity  is  a  relative  thing,  or,  in  other  words,  that  different 
animals  have  different  degrees  of  resistance  or  non-susceptibility 
to  toxic  bodies.  In  every  case  a  certain  dose  must  be  reached 
before  effects  can  be  observed,  and  up  to  that  point  the  animal 
has  resistance.  This  natural  resistance  is  found  to  present  very 
remarkable  degrees  of  variation  in  different  animals.  The  great 
resistance  of  the  common  fowl  to  the  toxin  of  the  tetanus  bacillus 
may  be  here  mentioned  (vide  p.  425),  and  large  amounts  of  this 
poison  can  be  injected  into  the  scorpion  without  producing  any 
effects  whatever ;  the  high  resistance  of  the  pigeon  to  morphia 
is  a  striking  example  in  the  case  of  vegetable  poisons.  This 
variation  in  resistance  to  toxins  applies  also  to  those  which 
produce  local  effects,  as  well  as  to  those  which  cause  symptoms 
of  general  poisoning.  Instances  of  this  are  furnished,  for 
example,  by  the  vegetable  poisons  ricin  and  abrin,  by  the  snake 
poisons,  and  by  bacterial  toxins  such  as  that  of  diphtheria.  We 
must  take  this  natural  resistance  for  granted,  though  it  is 
possible  that  ere  long  it  will  be  explained. 

According  to  Ehrlich's  view  of  the  constitution  of  toxins,  it 
might  be  due  to  the  want  of  combining  affinity  between  the 
tissue  cells  and  the  haptophorous  group  of  the  toxin ;  or,  on  the 
other  hand,  supposing  this  affinity  to  exist,  it  might  be  due  to 
an  innate  non-susceptibility  to  the  action  of  the  toxophorous 
group.  Certain  investigations  have  been  made  in  order  to 
determine  the  combining  affinity  of  the  nervous  system  of  the 
fowl  with  tetanus  toxin,  as  compared  with  that  obtaining  in  a 
susceptible  animal,  but  the  results  have  been  somewhat  contra- 
dictory. Accordingly,  a  general  statement  on  this  point  cannot 
at  present  be  made,  though  in  all  probability  variations  in  the 
susceptibility  to  the  toxophorous  group  will  be  found  to  play  a 
very  important  part.  It  has  been  shown  by  Muir  and  Browning 
by  means  of  haemolytic  tests  that  the  toxic  activity  of  complement, 
after  it  has  been  fixed  to  the  corpuscles,  varies  very  much ;  in  some 
instances  an  amount  of  complement,  which  would  rapidly  produce 
complete  lysis  of  one  kind  of  corpuscle,  may  have  practically  no 
effect  on  another,  even  though  it  enters  into  combination.  These 
results  are  of  importance  in  demonstrating  how  the  corresponding 
molecules  of  different  animals  may  vary  in  sensitiveness  to  toxic 
action. 

•Super sensitiveness  or  Anaphylaxis. 

Under  this  heading  are  to  be  grouped  a  number  of  phenomena 
which  in  their  character  and  results  afford  a  striking  contrast  to 
the  state  of  immunity.  The  common  feature  is  that  repeated  in 


BUPERSENSinVENESS  OR  ANAPHYLAXLS      559 

jections  of  certain  substances  in  sub-toxic  or  non-toxic  doses, — a 
suitable  interval  of  time  elapsing  between  the  injections, — may  be 
followed  by  markedly  toxic  or  even  fatal  symptoms,  and  a  similar 
result  may  follow  repeated  injectionsof  substances  which  are  practic- 
ally non-toxic  in  a  single  dose.  In  such  cases,  then,  a  condition  of 
sii]  >LTsensitiveness  to  the  particular  substance  has  been  established. 
The  substances  which  have  been  found  to  have  the  property  of 
calling  forth  this  condition  are  of  various  kinds,  including 
bacteria  and  their  toxins,  animal  poisons,  and  a  great  many 
foreign  proteins,  e.g.  those  of  serum,  milk,  egg  albumin,  etc.,  and 
it  is  to  be  noted  that  they  belong  to  the  group  of  substances 
which  can  act  as  antigens.  Probably  no  body  of  known  chemical 
constitution  develops  supersensitiveness ;  and,  just  as  tolerance, 
say  to  drugs,  is  to  be  distinguished  from  immunity,  so  ac- 
cumulative action  is  to  be  distinguished  from  supersensitiveness. 
Of  the  latter  condition  the  earliest  example  observed  was 
probably  the  special  susceptibility  of  tubercular  patients  to  the 
action  of  tuberculin,  to  which  reference  has  already  been  made 
(p.  284).  At  a  comparatively  early  date  also  it  was  found,  in 
the  case  of  diphtheria  and  tetanus  toxins,  that  in  certain 
instances  the  injection  of  a  minute  dose  followed  by  another  at 
a  suitable  interval  might  be  attended  by  serious  results;  and 
that  this  was  not  an  example  of  accumulative  action,  was  shown 
by  the  fact  that  the  sum  of  the  doses  might  amount  to  only 
a  fraction  of  a  lethal  dose.  Richet  investigated  a  similar 
phenomenon  in  the  case  of  a  toxic  substance  obtained  from  the 
tentacles  of  actiniae,  to  which  from  its  action  he  gave  the  name 
of  "congestin."  He  found  that  a  certain  time-interval  between 
the  injections  was  necessary ;  that  after  the  second  injection  the 
symptoms  occurred  with  remarkable  suddenness,  and  that  they 
appeared  to  be  practically  independent  of  the  size  of  the  first 
dose.  He  applied  the  term  anaphylaxis  to  the  supersensitive 
condition,  and  this  has  passed  into  general  use ;  he  found  also 
that  the  condition  lasted  several  weeks.  Arthus  found  that 
after  repeated  injections  of  horse  serum  in  rabbits  a  stage  was 
reached  at  which  an  additional  subcutaneous  injection  produced 
marked  oedema  and  even  necrosis,  while  an  intravenous  injection, 
harmless  to  an  untreated  animal,  brought  about  a  fatal  result. 
The  period  of  active  research  on  the  subject,  however,  may  be 
said  to  date  from  the  discovery  of  what  is  now  known  as  the 
"  phenomenon  of  Theobald  Smith."  This  observer  found  that 
guinea-pigs  which  had  been  treated  with  a  neutral  mixture  of 
diphtheria  toxin  and  antitoxin  might,  after  a  certain  interval  of 
time,  succumb  on  being  injected  with  a  quantity  of  normal  horse 


560  IMMUNITY 

serum.  It  was  afterwards  shown — especially  by  the  researches  of 
Otto  and  of  Rosenau  and  Anderson — that  the  sensitising  agent 
had  really  nothing  to  do  with  the  toxin  or  antitoxin,  but  was 
contained  in  the  normal  serum. 

After  this  brief  review  we  may  consider  some  of  the 
phenomena  of  serum  anaphylaxis,  as  it  is  now  called.  In  its 
study  horse  serum  has  been  chiefly  employed,  but  other  sera  are 
also  efficient,  and  guinea-pigs  are  the  most  suitable  test  animals, 
though  rabbits  have  also  been  used ;  in  the  case  of  mice  it  is 
difficult  if  not  impossible  to  bring  about  serum  anaphylaxis. 
There  is  first  of  all  the  sensitising  injection;  a  guinea-pig  is 
injected  subcutaneously  with  a  minute  quantity,  e.g.  '001  c.c.  of 
horse  serum,  though  even  smaller  amounts  may  be  sufficient  and 
other  methods  of  injection  may  also  be  employed.  After  a 
certain  number  of  days,  usually  twelve  as  a  minimum,  anaphylaxis 
has  been  established,  and  the  test  for  this  is  usually  made  by 
injecting  subcutaneously  5  c.c.  of  horse  serum.  In  the  ana- 
phylactic animal  severe  symptoms  occur ;  restlessness  and  hyper- 
algesia  are  followed  by  evidence  of  collapse,  the  temperature  falls 
markedly,  the  heart's  action  becomes  weak  and  the  respiration 
embarrassed ;  finally  death  may  occur.  The  intravenous  injec- 
tion of  a  smaller  amount  of  serum  brings  about  the  same  result 
more  rapidly.  It  is  to  be  noted  that  anaphylaxis  has  the 
character  of  specificity,  apparently  within  corresponding  limits 
to  immunity  (p.  521);  that  is,  it  is  manifested  only  on  the  re- 
injection  of  the  same  protein  substance  as  that  used  in  the  first 
instance.  There  is  also  a  passive  anaphylaxis,  as  is  shown  by 
the  fact  that  if  a  certain  amount  of  the  serum  of  an  anaphylactic 
guinea-pig  be  injected  into  a  normal  one,  the  latter  becomes  ana- 
phylactic, so  that  the  characteristic  symptoms  appear  in  it  when 
the  test  amount  of  horse  serum  is  injected.  In  most  instances 
an  interval  of  about  twenty-four  hours  must,  however,  elapse 
between  the  injections  (Otto)  ;  if  the  two  injections  are  made 
at  the  same  time  there  is  usually  no  result.  Another  interesting 
observation  has  been  made,  namely,  that  the  young  of  anaphylactic 
mothers  may  also  be  anaphylactic,  and  the  condition  may  last 
for  some  time  after  birth.  It  is  also  possible  to  produce  a 
condition  of  anti-anaphylaxis,  that  is,  to  vaccinate  against  the 
supersensitive  condition.  If,  for  example,  the  sensitising  dose  of 
horse  serum  is  injected,  and  then  before  anaphylaxis  is  established 
(i.e.  sometime  before  the  twelfth  day)  another  injection  of  a 
considerable  quantity  is  made,  anaphylaxis  does  not  appear,  and 
the  animal  is  non-susceptible  to  further  injections  of  small  closes 
for  a  considerable  period  of  time. 


SUPERSENSITIVENESS  OR  ANAPHYLAXIS      561 

With  regard  to  the  mechanism  underlying  the  phenomena 
described,  practically  all  observers  are  agreed  that  there  is  a 
profound  affection  of  the  nervous  system ;  but  it  is  still  an  open 
question  as  to  \\lirtlnT  the  severe  and  practically  simultaneous 
affections  of  the  other  systems  are  merely  secondary,  or  whether 
they  are  independently  produced  by  some  change  common  to 
all.  A  great  fall  in  the  blood-pressure  is  an  important 
plu'iiomenon,  and  is  due  chiefly  to  a  general  vaso-dilatation ; 
and  it  has  been  pointed  out  by  Auer  and  Lewis  that  in  the 
case  of  guinea-pigs  there  occurs  a  spasm  of  the  muscle  fibres  in 
the  fine  bronchi  and  alveolar  passages,  the  chest-wall  being  fixed 
in  full  inspiration  at  the  time  of  death.  Besredka  has  shown 
that  the  fatal  symptoms  are  more  rapidly  produced  in  an 
anaphylactic  animal  and  with  a  smaller  dose  of  serum,  when 
the  injection  is  made  directly  into  the  brain,  than  by  any  other 
method.  Further,  seeing  that  a  single  dose  of  horse  serum  is  not 
toxic  to  the  guinea-pig,  and  that  an  interval  of  several  days 
must  elapse  before  anaphylaxis  is  established,  the  majority  of 
observers  consider  that  at  least  two  substances  are  concerned, 
one  of  which  is  contained  in  normal  horse  serum,  whilst  the 
other  is  developed  in  the  guinea-pig  in  response  to  the  presence 
of  the  first  or  of  some  other  substance  after  the  manner  of  an 
anti-substance.  To  this  newly  developed  substance  the  name 
of  "  anaphylactic  reaction-body  "  is  often  given.  The  phenomena 
thus  depend  upon  the  co-operation  of  the  reaction-body  with  a 
substance  or  substances  in  the  horse  serum,  and  a  rapid  union 
of  the  two,  probably  within  the  nerve-cells,  brings  about  the 
anaphylactic  shock.  Passive  anaphylaxis  would  thus  be  due  to 
the  transference  of  the  reaction-body  to  a  fresh  animal,  and  the 
interval  necessary  before  the  second  injection  might  depend 
upon  the  time  required  for  the  reaction-body  to  accumulate  in 
.sufficient  quantity  within  the  nerve-cells. 

lU'sivdka  considers  that  the  sensitising  and  the  toxic  factors 
in  the  horse  serum  are  not  one  and  the  same.  He  finds  that 
serum  heated  to  a  certain  temperature  may  still  have  the  power 
of  inducing  the  condition  of  anaphylaxis,  but  has  lost  the  power 
of  bringing  about  the  toxic  phenomena  when  injected  into  an 
anaphylactic  animal.  Gay  and  Adler  similarly  find  that  the 
sensitising  substance  (anaphylactin)  is  contained  in  the 
i-ii^lobulin  fraction  of  the  serum  while  the  other  is  not. 
llrsrcdka  accordingly  puts  forward  the  view  that  in  the  horse 
si-rum  there  are  two  substances  or  rather  factors,  namely, 
sen8ifii/i*''it»!/'-/i.  which  is  thermostable,  and  anti-sensibilisin, 
which  is  thermolabile.  When  the  serum  is  injected  the  former 

36 


562  IMMUNITY 

gives  rise  to  sensibilisin  as  an  anti-substance  ;  and  when,  after 
a  suitable  time,  fresh  serum  is  injected,  the  anti-sensibilisin  com- 
bines with  the  sensibilisin,  and  thus  the  anaphylactic  shock 
results.  In  view,  however,  of  the  specific  nature  of  the 
phenomena,  it  would  appear  that  both  sensibilisinogen  and  anti- 
sensibilisin  must  have  the  same  special  combining  group  for 
sensibilisin,  and  it  is  accordingly  difficult  to  see  why  the  latter 
should  not  also  act  as  an  antigen.  He  has  also  found  that 
when  an  animal  is  anaesthetised  with  ether  the  anaphylactic 
shock  may  be  averted.  Other  workers  at  this  subject 
hold  that  there  are  only  two  substances  concerned,  and 
some  consider  that  the  phenomena  depends  on  a  process  of 
precipitation.  Friedberger,  for  example,  considers  that  the  real 
toxic  agent  is  formed  by  the  action  of  complement  on  serum- 
precipitate  (antigen  +  precipitin).  To  this  substance  he  gives  the 
name  "  anaphylatoxin,"  and  in  support  of  his  view  he  has  shown 
that  guinea-pig's  complement,  after  it  has  been  allowed  to 
act  for  some  time  on  such  a  precipitate  and  then  removed  by 
the  centrifuge,  has  acquired  toxic  properties,  and  produces  the 
symptoms  of  anaphylaxis  when  injected  into  a  normal  guinea 
pig.  He  also  points  out  that  during  anaphylactic  phenomena, 
especially  in  the  case  of  passive  anaphylaxis,  there  is  a  great 
fall  of  complement  in  the  blood  of  the  animal,  and  Scott  has 
brought  forward  facts  which  indicate  that  there  is  a  close 
relationship  between  this  fall  in  complement  and  the  occurrence 
of  the  symptoms  in  anaphylaxis.  On  the  other  hand,  Gay  and 
Southard  do  not  believe  in  the  theory  of  a  reaction-body. 
They  consider  that  the  condition  depends  on  the  presence  of  a 
substance  in  the  serum  which  they  call  anaphylactin,  and  which 
persists  in  the  blood  of  the  guinea-pig  for  a  long  period  of  time. 
This  acts  as  a  slight  irritant  to  the  nerve-cells,  and  produces  in 
them  an  increased  affinity  for  certain  molecules  in  the  serum. 
Accordingly,  when  the  second  injection  is  made,  the  rapid  com- 
bination of  these  molecules  with  the  cells  results  in  the  disturb- 
ances described.  This  view  has,  however,  received  little  support, 
and  there  are  various  facts  against  it,  especially  in  relation  to 
the  transference  of  anaphylaxis.  Others,  again,  e.g.  Citron, 
consider  that  supersensitiveness  is  so  closely  allied  to  immunity 
as  to  really  represent  the  earliest  stage  in  its  development.  At 
present  it  is  impossible  to  express  an  opinion  with  regard  to  the 
real  nature  of  the  phenomena.  Manifestly,  however,  if  they 
depend  upon  the  existence  of  an  anti-substance  in  the  nerve:cells 
and  the  cells  of  other  organs,  the  injection  of  fresh  serum,  before 
the  anti-substance  is  fully  formed,  say  on  the  ninth  day  after 


THE  SERUM  DISEASE  IN  MAN  563 

the  first  injection,  will  lead  to  its  combination  and  thus  to  its 
being  used  up,  and  thus  the  condition  of  anti-anaphylaxis  will 
be  established. 

It  is  still  an  open  question  as  to  what  extent  the  phenomena 
of  anaphylaxis  just  described  are  of  the  same  nature  as  the 
supersensitiveness  manifested  by  patients  suffering  from  disease 
to  the  products  of  the  corresponding  organism,  e.y.  to  tuberculin, 
mallein,  etc.  (pp.  284,  314)  ;  though  in  all  probability  they  are  at 
least  similar  in  essence.  It  was  held  for  some  time  as  a  distinc- 
tion that  this  s u pel-sensitiveness  in  infections  to  bacterial  products 
could  not  be  transferred  to  another  animal,  but  recent  observa- 
tions show  that  in  certain  circumstances  this  is  possible  in  the 
case  of  tuberculin.  There  is  no  doubt  that  the  supersensitive 
condition  must  play  an  important  part  in  the  clinical  manifesta- 
tions of  many  diseases.  For  example,  the  sensitiveness  of 
tubercular  patients  to  tuberculin  shows  that  the  symptoms  in 
this  disease  are  evidently  produced  by  the  absorption  from  the 
tubercular  foci  of  a  smaller  amount  of  toxin  than  would  be 
necessary  to  produce  effects  in  a  normal  individual.  And  the 
sensitiveness  of  the  conjunctiva  in  typhoid  fever  to  the  products 
of  the  bacillus  suggests  that  in  this  disease  also  supersensitive- 
ness plays  an  important  part.  It  is  also  possible  that  the 
repeated  absorption  of  proteins,  harmless  in  single  doses,  may 
I  fat  I  to  toxic  symptoms,  and  in  a  similar  way  may  possibly  be 
explained  the  relative  non-toxicity  of  the  products  of  certain 
bacteria  when  tested  in  the  usual  manner.  But  with  regard  to 
all  these  questions,  which  are  of  the  highest  importance,  much 
further  research  is  still  necessary. 

The  Serum  Disease  in  Man. — This  condition,  which  is 
intimately  related  to  suj>ersensitiveness,  includes  the  phenomena 
which  have  been  observed  after  the  injection  of  anti-diphtheric 
and  other  sera.  The  real  factor  is  the  introduction  of  foreign 
sera  into  the  human  tissues.  As  in  the  case  of  anaphylaxis,  as 
above  described,  there  is  here  also  a  period  of  incubation,  of  eight 
to  twelve  days  on  the  average  ;  after  which,  in  a  certain  proportion 
of  cases  (in  about  20  per  cent,  after  the  injection  of  a  fairly 
large  amount  of  horse  serum,  a  group  of  characteristic  symptoms 
appear.  There  may  be  as  prodromal  symptoms,  swelling  and 
tenderness  at  the  site  of  injection,  and  in  the  corresponding 
lymphatic  glands,  and  thereafter  general  exanthemata  appear. 
These  are  usually  of  an  urticarial  type,  but  may  be  erythematous 
or  morbilliform.  There  is  usually  moderate  pyrexia  of  a 
remittent  type,  and  sometimes  cedema  and  slight  albuminuria 
are  present ;  occasionally  there  are  pains  in  the  joints ;  there  is 


564  IMMUNITY 

also  often  leucopenia  due  to  a  fall  in  the  number  of  polymorpho- 
nuclear  leucocytes.  These  symptoms  last  for  a  few  days  and 
then  disappear.  Such  are  the  phenomena  of  the  serum  disease 
after  a  single  injection  of  the  foreign  serum.  There  are,  however, 
two  other  types  of  reaction  described  by  v.  Pirquet  and  Schick, 
namely,  the  immediate  and  the  accelerated  reactions.  The  im- 
mediate reaction  is  seen  when  a  large  dose  of  serum  has  been 
administered  and  then  after  a  certain  interval  of  time  another 
dose  of  serum  is  injected.  This  interval  is  usually  from  twelve 
days  to  eight  weeks,  though  sometimes  as  long  as  six  months. 
The  symptoms  of  the  immediate  reaction,  which  appear  shortly 
after  the  injection,  or  at  least  within  twenty-four  hours,  are  an 
intense  oedema  locally,  general  exanthemata  and  pyrexia,  though 
the  general  phenomena  are  often  little  marked.  The  symptoms 
pass  off  comparatively  quickly,  usually  within  twenty-four  hours. 
The  accelerated  reaction  is  also  seen  after  a  second  injection, 
and  it  may  occur  from  six  weeks  up  to  many  months  after  the 
first  injection.  In  the  case  of  the  accelerated  reaction  there  is 
an  incubation  period,  but  it  is  shorter  than  in  the  case  of  the 
first  injection,  being  usually  five  to  seven  days ;  the  symptoms 
resemble  those  in  the  ordinary  reaction  as  described  above,  but 
are  of  rather  more  acute  onset  and  last  a  shorter  time.  In  the 
interval  from  about  the  sixth  week  to  the  sixth  month,  there 
may  occur  both  the  immediate  reaction,  and  also  a  few  days 
later  an  accelerated  reaction. 

The  nature  of  the  serum  disease  is  not  yet  fully  understood, 
but  in  all  probability  depends  upon  the  development  of  a  reaction- 
body  or  anti-substance,  and  the  combination  of  this  with  a  sub- 
stance in  the  serum,  probably  an  antigen,  leads  to  the  symptoms. 
We  suppose  that  the  substances  in  the  serum  gradually  disappear 
from  the  body  after  the  injection  ;  from  about  the  eighth  day 
onward  anti-substances  appear  in  the  blood  in  large  amount, 
and  if  antigens  are  still  present,  the  combination  of  the  two 
brings  about  the  phenomena  described.  Manifestly,  if  the 
antigens  have  disappeared  before  the  anti-substances  appear  in 
quantity,  there  will  be  no  symptoms.  At  a  later  period  anti- 
substances  will  be  present  alone  in  the  serum,  and  then  the 
injection  of  fresh  antigens  brings  about  an  immediate  reaction. 
After  the  anti-substances  have  disappeared,  the  injection  of 
fresh  serum  causes  no  immediate  reaction,  but  the  mechanism 
of  reaction  has  been  stimulated  by  the  first  injection ;  anti- 
substances  thus  appear  more  quickly  after  the  second  injection, 
hence  the  reaction  is  accelerated  as  compared  with  the  reaction 
after  the  first  injection. 


APPENDIX  A. 

SMALLPOX  AND  VACCINATION. 

SMALLPOX  is  a  disease  to  which  much  study  has  been  devoted, 
owing,  on  the  one  hand,  to  the  havoc  which  it  formerly  wrought 
among  the  nations  of  Europe, — a  havoc  which  at  the  present 
day  it  is  difficult  to  realise, — and,  on  the  other  hand,  to  the 
controversies  which  have  arisen  in  connection  with  the  active 
immunisation  against  it  introduced  by  Jenner.  Though  there 
is  little  doubt  that  a  contagium  vivum  is  concerned  in  its 
occurrence,  the  etiological  relationship  of  any  particular  organ- 
ism to  smallpox  has  still  to  be  proved ;  and  with  regard  to 
Jennerian  vaccination,  it  is  only  the  advance  of  bacteriological 
knowledge  which  is  now  enabling  us  to  understand  the  prin- 
ciples which  underlie  the  treatment,  and  which  is  furnishing 
methods  whereby  the  vexed  questions  concerned  will  probably 
be  satisfactorily  settled.  We  cannot  here  do  more  than  touch 
on  some  of  the  results  of  investigation  with  regard  to  the 
disease. 

Jennerian  Vaccination. — Up  to  Jenner's  time  the  only 
means  adopted  to  mitigate  the  disease  had  been  by  inoculation 
(by  scarification)  of  virus  taken  from  a  smallpox  pustule, 
especially  from  a  mild  case.  By  this  means  it  was  shown  that 
in  the  great  majority  of  cases  a  mild  form  of  the  disease  was 
originated.  It  had  previously  been  known  that  one  attack  of 
the  disease  protected  against  future  infection,  and  that  the  mild 
attack  produced  by  inoculation  also  had  this  effect.  This 
inoculation  method  had  long  been  practised  in  various  parts  of 
the  world,  and  had  considerable  popularity  all  over  Europe 
during  the  eighteenth  century.  Its  disadvantage  was  that  the 
resulting  disease,  though  mild,  was  still  infectious,  and  thus 
might  be  the  starting-point  of  a  virulent  form  among  un- 
protected persons.  Jenner's  discovery  was  published  when 
inoculation  was  still  considerably  practised.  It  was  founded  on 
the  popular  belief  that  those  who  had  contracted  cowpox  from 

565 


566  SMALLPOX  AND  VACCINATION 

an  affected  animal  were  insusceptible  to  subsequent  infection 
from  smallpox.  In  the  horse  there  occurs  a  disease  known  as 
horsepox,  especially  tending  .to  arise  in  wet,  cold  springs,  which 
consists  in  an  inflammatory  condition  about  the  hocks,  giving 
rise  to  ulceration.  Jenner  believed  that  the  matter  from  these 
ulcers,  when  transferred  by  the  hands  of  men  who  dressed  the 
sores  to  the  teats  of  cows  subsequently  milked  by  them,  gave 
rise  to  cowpox  in  the  latter.  This  disease  was  thus  identical 
with  horsepox  in  epidemics  of  which  it  had  its  origin.  Jenner 
was,  however,  probably  in  error  in  confounding  horsepox  with 
another  disease  of  horses,  namely,  grease.  Cowpox  manifests 
itself  as  a  papular  eruption  on  the  teats ;  the  papules  become 
pustules ;  their  contents  dry  up  to  form  scabs,  or  more  or  less 
deep  ulcers  occur  at  their  sites.  From  such  a  lesion  the  hands 
of  the  milkers  may  become  infected  through  abrasions,  and  a 
similar  local  eruption  occurs,  with  general  symptoms  in  the 
form  of  slight  fever,  malaise,  and  loss  of  appetite.  It  is  this 
illness  which,  according  to  Jenner,  gives  rise  to  immunity  from 
smallpox  infection.  He  showed  experimentally  that  persons 
who  had  suffered  from  such  attacks  did  not  react  to  inoculation 
with  smallpox ;  and  further,  that  persons  to  whom  he  communi- 
cated cowpox  artificially  were  similarly  immune.  The  results 
of  Jenner's  observations  and  experiments  were  published  in  1798 
under  the  title,  An  Inquiry  into  the  Causes  and  Effects  of  the 
Variola  Vaccince.  Though  from  the  first  Jennerian  vaccina- 
tion had  many  opponents,  it  gradually  gained  the  confidence  of 
the  unprejudiced,  and  became  extensively  practised  all  over  the 
world,  as  it  is  at  the  present  day. 

The  evidence  in  favour  of  vaccination  is  very  strong.  There 
is  no  doubt  that  inoculation  with  lymph  properly  taken  from  a 
case  of  cowpox,  can  be  maintained  with  very  little  variation  in 
strength  for  a  long  time  by  passage  from  calf  to  calf,  and  such 
calves  are  now  the  usual  source  of  the  lymph  used  for  human 
vaccination.  When  lymph  derived  from  them  is  used  for  the 
latter  purpose,  immunity  against  smallpox  is  conferred  on  the 
vaccinated  individual.  It  has  been  objected  that  some  of 
the  lymph  which  has  been  used  has  been  derived  from  calves 
inoculated,  not  with  cowpox,  but  with  human  smallpox.  It  is 
possible  that  this  may  have  occurred  in  some  of  the  strains  of 
lymph  in  use  shortly  after  the  publication  of  Jenner's  discovery, 
but  most  of  the  strains  at  present  in  use  have  probably  been 
derived  originally  from  cowpox.  The  most  striking  evidence  in 
favour  of  vaccination  is  derived  from 'its  effects  among  the  staffs 
of  smallpox  hospitals,  for  here,  in  numerous  instances,  it  is  only 


RELATIONSHIP  OF  SMALLPOX  TO  COWPOX     567 

the  unvaccinated  individuals  who  have  contracted  the  disease. 
While  vaccination  is  undoubtedly  efficacious  in  protecting  against 
smallpox,  Jenner  was  wrong  in  supposing  that  a  vaccination  in 
infancy  afforded  protection  for  more  than  a  certain  number  of 
years  thereafter.  It  has  been  noted  in  smallpox  epidemics 
which  1 1  avo  occurred  since  the  introduction  of  vaccination,  that 
whereas  young  unprotected  subjects  readily  contract  the  disease, 
those  vaccinated  as  infants  escape  more  or  less  till  after  the 
thirteenth  to  the  fifteenth  years.  It  has  become,  therefore,  more 
and  more  evident  that  revaccination  is  necessary  if  immunity  is 
to  continue  ;  and  where  this  is  done  in  any  population,  smallpox 
becomes  a  rare  disease,  as  has  happened  in  the  German  army, 
where  the  mortality  is  practically  nil.  The  whole  question  of 
the  efficacy  of  vaccination  was  investigated  in  this  country  in 
1896  by  a  Royal  Commission,  whose  general  conclusions  were 
as  follows : — Vaccination  diminishes  the  liability  to  attack  by 
smallpox,  and  when  the  latter  does  occur,  the  disease  is  milder 
and  less  fatal.  Protection  against  attack  is  greatest  during 
nine  or  ten  years  after  vaccination.  It  is  still  efficacious  for  a 
further  j>eriod  of  five  years,  and  possibly  never  wholly  ceases. 
The  power  of  vaccination  to  modify  an  attack  outlasts  its  power 
wholly  to  wrard  it  off.  Revaccination  restores  protection,  but 
this  operation  must  be  from  time  to  time  repeated.  Vaccination 
is  beneficial  according  to  the  thoroughness  with  which  it  is 
performed 

The  Relationship  of  Smallpox  (Variola)  to  Cowpox 
(Vaccinia). — This  is  the  question  regarding  which,  since  the 
introduction  of  vaccination  the  greatest  controversy  has  taken 
place ;  a  subsidiary  point  has  been  the  inter-relationships  within 
the  group  of  animal  diseases  which  includes  cowpox,  horsepox, 
sheep-pox,  and  cattle-plague.  With  reference  to  smallpox  and 
cowpox  the  problem  has  been,  Are  they  identical  or  not  1  There 
is  no  doubt  that  cowpox  can  be  communicated  to  man,  in  whom 
it  produces  the  eruption  limited  to  the  point  of  inoculation,  and 
the  slight  general  symptoms  which  vaccination  with  calf  lymph 
has  made  familiar.  Apparently  against  the  view  that  cowpox 
is  a  modified  smallpox  are  the  facts  that  it  never  reproduces  in 
man  a  general  eruption,  and  that  the  local  eruption  is  only 
infectious  when  matter  from  it  is  introduced  into  an  abrasion. 
The  loss  of  infectiveness  by  transmission  through  the  body  of  a 
relatively  insusceptible  animal  is  a  condition  of  which  we  have 
already  seen  many  instances  in  other  diseases,  and  the  uniformity 
of  the  type  of  the  affection  resulting  from  vaccination  with  calf 
lymph  finds  a  parallel  in  such  a  disease  as  hydrophobia,  where, 


568  SMALLPOX  AND  VACCINATION 

after  passage  through  a  series  of  monkeys,  a  virus  of  attenuated 
but  constant  virulence  can  be  obtained.  We  have  seen  there 
are  good  grounds  for  believing  that  the  virus  of  calf  lymph 
confers  immunity  against  human  smallpox.  In  considering  the 
relationships  of  cowpox  and  smallpox,  this  is  an  important 
though  subsidiary  point ;  for  at  present  it  is  questionable 
whether  there  are  any  well-authenticated  instances  of  one 
disease  having  the  capacity  of  conferring  immunity  against 
another.  The  most  difficult  question  in  this  connection  is  what 
happens  when  inoculations  of  smallpox  matter  are  made  on 
cattle.  Chauveau  denies  that  in  such  circumstances  cowpox  is 
obtained.  He,  however,  only  experimented  on  adult  cows.  The 
transformation  has  been  accomplished  by  many  observers, 
including,  in  this  country,  Simpson,  Klein,  Hime,  and  Copeman. 
The  general  result  of  these  experiments  has  been  that  if  a  series 
of  calves  is  inoculated  with  variolous  matter,  in  the  first  there 
may  not  be  much  local  reaction,  though  redness  and  swelling 
appear  at  the  point  of  inoculation,  and  some  general  symptoms 
manifest  'themselves.  On  squeezing  some  of  the  lymph  from 
such  lesion  as  occurs,  and  using  it  to  continue  the  passages 
through  other  calves,  after  a  very  few  transfers  a  local  reaction 
indistinguishable  from  that  caused  by  cowpox  lymph  generally 
takes  place,  and  the  animals  are  now  found  to  be  immune 
against  the  latter.  Not  only  so,  but  on  using  for  human 
vaccination  the  lymph  from  such  variolated  calves,  results 
indistinguishable  from  those  produced  by  vaccine  lymph  are 
obtained,  and  the  transitory  illness  which  follows,  unlike  that 
produced  in  man  by  inoculation  with  smallpox  lymph,  is  no 
longer  infectious.  In  fact,  many  of  the  strains  of  lymph  in  use 
in  Germany  at  present  have  been  derived  thus  from  the  variola- 
tion  of  calves.  The  criticism  of  these  experiments  which  has 
been  offered,  namely,  that  since  many  of  them  were  performed 
in  vaccine  establishments,  the  calves  were  probably  at  the  same 
time  infected  with  vaccinia,  is  not  of  great  weight,  as  in  all  the 
recent  cases  at  least,  very  elaborate  precautions  have  been 
adopted  against  such  a  contingency.  And  at  any  rate  it 
would  be  rather  extraordinary  that  this  accident  should  happen 
in  every  case.  We  can,  therefore,  say  that  at  present  there 
is  the  very  strongest  ground  for  holding  not  only  that  vaccinia 
confers  immunity  against  variola,  but  that  variola  confers 
immunity  against  vaccinia.  The  experimentum  crucis-  for 
establishing  the  identity  of  the  two  diseases  would  of  course 
be  the  isolation  of  the  same  micro-organism  from  both,  and  the 
obtaining  of  all  the  results  just  detailed  by  means  of  pure 


BACTERIA  IN  SMALLPOX  '  569 

cultures  or  the  products  of  such.  In  the  absence  of  this 
evidence  we  are  at  present  justified  in  considering  that  there  is 
strong  reason  for  believing  that  vaccinia  and  variola  are  the  same 
disease,  and  that  the  differences  between  them  result  from  the 
relative  susceptibilities  of  the  two  species  of  animals  in  which 
they  occur  naturally. 

With  regard  to  the  relation  of  cowpox  to  horsepox,  it  is 
extremely  probable  that  they  are  the  same  disease.  Some 
epidemics  of  the  former  have  originated  from  the  horse,  but  in 
other  cases  such  a  source  has  not  been  traced.  Cattle  plague 
fnmi  the  clinical  standpoint,  and  also  from  that  of  pathological 
anatomy,  resembles  very  closely  human  smallpox.  Though 
each  of  the  two  diseases  is  extremely  infectious  to  its  appropriate 
animal,  there  is  no  record  of  cattle-plague  giving  rise  to  small- 
pox in  man  or  vice  versa.  When  matter  from  a  cattle-plague 
pustule  is  inoculated  in  man,  a  pustule  resembling  a  vaccine 
pustule  occurs,  and  further,  the  individual  is  asserted  to  be  now 
immune  to  vaccination ;  but  vaccination  of  cattle  with  cowpox 
lymph  offers  no  protection  against  cattle-plague,  though  some 
have  looked  on  the  latter  as  merely  a  malignant  cowpox.  Sheep- 
pox  also  has  many  clinical  and  pathological  analogies  with 
human  smallpox,  and  facts  as  to  its  relation  to  cowpox  vaccina- 
tion similar  to  those  observed  in  cattle-plague  have  been 
reported.  Smallpox,  cowpox,  cattle-plague,  horsepox,  and  sheep- 
pox,  in  short,  constitute  an  interesting  group  of  analogous 
diseases,  of  the  true  relationships  of  which  to  one  another  we 
are,  however,  still  ignorant. 

Micro-organisms  associated  with  Smallpox. — Burdon  Sander- 
son and  other  observers  early  pointed  out  that  in  matter  derived 
from  variolous  and  vaccine  pustules  (especially  the  later  stages 
of  the  latter),  pyogenic  organisms  are  always  present,  e.g. 
mtaphylococcuz  aureus  and  staphylococcus  cereus  Jlavus,  and  many 
of  the  ordinary  skin  saprophytes  also  are  often  present,  but  no 
organism  has  ever  been  isolated  which  on  transference  to  animals 
lias  been  shown  to  have  any  specific  relationship  to  the  disease. 
Streptococci  have  also  been  described  as  agglutinable  by  the  sera 
of  smallpox  patients  and  of  vaccinated  persons ;  such  sera,  it  may 
be  said,  had  no  effect  on  other  strains  of  streptococci.  Calmette 
and  Guerin  have  described  very  minute  granules  in  the  lymph 
\\hirli  could  not  be  cultivated,  but  which  persisted  after  all  the 
bacteria  had  been  removed.  (The  method  by  which  the  latter 
was  accomplished  was  by  exciting  a  leucocytosis  in  a  rabbit's 
l>eritoneum  and  then  introducing  the  vaccinal  lymph ;  the 
leucocytes  phagocyted  the  bacteria  so  that  the  lymph  no  longer 


570  SMALLPOX  AND  VACCINATION 

gave  cultures  on  ordinary  media.     It  was,  however,  still  potent 
to  produce  vaccinia.) 

Klein  and  also,  independently,  Copeman,  have  observed  an  organism 
in  lymph  taken  from  a  vaccine  pustule  in  a  calf  on  the  fifth  and  sixth 
days,  in  human  vaccine  lymph  on  the  eighth  day,  and  in  lymph  from 
a  smallpox  pustule  on  the  fourth  day.  To  demonstrate  the  bacilli, 
cover-glass  films  are  dried  and  placed  for  five  minutes  in  acetic  acid  (1 
in  2),  washed  in  distilled  water,  dried,  and  placed  in  alcoholic  gentian- 
violet  for  from  twenty-four  to  forty-eight  hours,  after  which  they  are 
washed  in  water  and  mounted.  Copeman  and  Kent  also  found  the 
bacilli  in  sections  of  vaccine  pustules  stained  by  Loffler's  methylene-blue, 
or  by  Gram's  method.  The  organisms  are  *4  to  '8  /UL  in  length,  and 
one-third  to  a  half  of  this  in  thickness.  They  are  generally  thinner  and 
stain  better  at  the  ends  than  at  the  middle.  They  occur  in  groups  of 
from  three  to  ten  in  both  the  lymph  and  the  tissues.  In  the  centre  of 
their  protoplasm  there  is  often  a  clear  globule,  which  is  looked  on  as  a 
spore.  They  have  hitherto  resisted  the  ordinary  isolation  methods,  a 
fact  which  is  rather  in  favour  of  their  non-saprophytic  nature.  By 
inoculating  fresh  eggs  with  the  crusts  of  smallpox  pustules  Copeman.  has, 
however,  obtained  a  growth  of  a  bacillus  resembling  that  found  by  him 
in  the  tissues.  Though  subcultures  on  ordinary  media  have  been 
obtained,  the  pathogenic  effects  of  these  have  not  been  fully  investigated, 
and  thus  the  identity  of  this  bacillus  with  that  seen  in  the  tissues  is 
not  proved. 

Various  observers  have  described  structures  in  the  epithelial 
cells  in  the  neighbourhood  of  the  smallpox  or  vaccine  pustules, 
which  they  have  interpreted  as  being  protozoa.  Thus  Ruffer 
and  Plimmer  describe  as  occurring  in  clear  vacuoles  in  the  cells 
of  the  rete  Malpighii  at  the  edge  of  the  pustule  (in  paraffin 
sections  of  vaccine  and  smallpox  pustules  carefully  hardened  in 
alcohol,  and  stained  by  the  Ehrlich-Biondi  mixture)  small  round 
bodies  of  about  four  times  the  size  of  a  staphylococcus  pyogenes, 
coloured  red  by  the  acid  fuchsin,  sometimes  with  a  central  part 
stained  by  the  methyl-green.  These  are  described  as  multiplying 
by  simple  division,  and  in  the  living  condition  exhibiting 
amoeboid  movement.  Similar  bodies  have  been  described  by 
Reed  in  the  blood  of  smallpox  patients  and  of  vaccinated 
children  and  calves. 

These  are  probably  the  bodies  described  by  Guarnieri  and  to 
which  considerable  attention  has  been  paid.  They  are  from 
1  to  8  /A  in  diameter,  are  round,  oval,  or  sickle-shaped,  and  stain 
by  ordinary  nuclear  dyes.  They  lie  in  the  cells  in  spaces 
often  near  the  nucleus,  and  are  readily  demonstrable  in  vaccine 
pustules  and  also  in  the  experimental  lesions  which  can  be 
produced  in  the  rabbit's  cornea,  the  larger  bodies  being  denned 
in  the  cells  towards  the  centre  of  the  lesion.  These  bodies 


NATURE  OF  VACCINATION"  571 

have  been  looked  on  by  many  as  protozoa,  and  Guarnieri  himself 
stated  that  multiplication  could  be  seen  occurring  in  them  in 
fresh  lymph,  but  Ewing  and  also  Prowazek  have  brought  forward 
strong  evidence  for  the  appearances  being  due  to  nuclear  changes, 
though  the  latter  observer  considers  them  to  be  the  effect  of  a 
specific  reaction  of  epithelial  cells  against  the  variolous  virus. 
Here  it  may  be  said  Wasielewski  has  shown  that  they  persist 
through  46  transfers  on  the  cornea  of  the  rabbit  and,  further,  no 
similar  appearances  have  been  found  in  other  skin  lesions. 
Prowazek  examined  material  fixed  in  a  hot  mixture  of  two- 
thirds  saturated  perchloride  of  mercury  and  one-third  98  per 
cent,  alcohol,  washed  in  40  per  cent,  iodine  alcohol  and  stained 
in  Grenacher's  ha3matoxylin,  and  found  bodies  in  the  epithelial 
cells  1  to  4  /u,  in  size,  sharply  contoured  and  having  ragged 
edges  as  if  made  up  of  massed  chromosomes.  These  were  often 
broader  at  one  end  than  at  the  other,  and  appearances  have 
been  seen  which  suggest  longitudinal  division.  Prowazek  has 
also  seen  these  "  lymph-bodies,"  as  he  has  called  them,  in  the 
lymph,  and  he  inclines  to  the  idea  that  they  may  be  protozoa. 
Bonhoff  and  also  Carini  have  described  spirochsetes  as  occurring 
in  variolous  lesions,  but  this  has  not  been  confirmed.  Volpino 
states  that  in  the  epithelial  cells  in  corneal  infection  in  rabbits, 
minute  motile  bodies  can  be  discerned  which  do  not  occur  in 
other  corneal  inflammations.  Future  investigations  must  show 
what  significance  is  to  be  attached  to  these  various  observations. 

The  causal  organism  of  smallpox  is  probably  very  small,  as, 
though  there  has  been  some  difference  in  opinion  on  this  point, 
there  is  little  doubt  that  it  will  pass  through  the  coarser  porcelain 
filters. 

The  Nature  of  Vaccination. — From  the  facts  known  regarding 
vaccination  we  are  justified  in  supposing  that  the  principle 
underlying  the  efficacy  of  this  prophylactic  is  the  establishment 
of  an  active  immunity  against  the  causal  organism,  which  is 
sufficiently  lasting  to  protect  the  vaccinated  individual  for  a 
considerable  time.  Although  the  virus  of  smallpox  is  unknown, 
several  attempts  have  been  made  by  indirect  methods  to 
establish  the  existence  of  reactions  similar  to  those  occurring 
in  other  immunisations.  Thus,  in  cases  of  human  smallpox 
and  in  animals  intravenously  injected  with  the  vaccine  lymph,  it 
is  stated  that  the  serum  when  mixed  with  vaccine  lymph  acquires 
the  property  of  deviating  complement,  and  evidence  has  also  been 
obtained  by  Prowazek  that  the  serum  of  monkeys  infected 
subcutaneously  contains  substances  of  the  nature  of  anti-bodies, 
for,  when  it  is  mixed  with  the  lymph,  the  mixture  is  not 


572  SMALLPOX  AND  VACCINATION 

capable  of  originating  a  vaccine  pustule  in  children.     Phenomena 
of  hypersensitiveness  on  revaccination  have  also  been  described. 

Considerable  attention  has  been  devoted  to  the  study  of  the 
effects  of  corneal  and  cutaneous  infection  in  the  rabbit  and 
monkey.  Here  it  has  been  found  that  the  infection  of  one 
cornea  protects  that  eye  against  re-inoculation  but  not  the  other 
eye.  Further,  it  is  stated  that  while  cutaneous  vaccination 
causes  the  general  skin  surface  after  about  ten  days  to  become 
insusceptible,  the  cornea  may  still  in  the  monkey  be  sensitive 
(this  last  fact  is  said  not  to  be  true  for  the  rabbit).  Again, 
intraperitoneal  infection  with  lymph  is  said  not  to  be  followed 
by  cutaneous  immunity.  Such  facts  have  led  some  to  suppose 
that  smallpox  is  essentially  a  disease  of  the  cutaneous  tissues. 
In  it  we  would  have  another  example  of  local  infection  such  as 
is  found  in  tubercular  leprosy,  lupus,  and  certain  other  skin 
infections.  Prowazek  strongly  holds  that  in  cutaneous  vaccinal 
infection  there  is  never  a  distribution  of  the  virus  throughout 
the  organs,  but  this  result  has  been  disputed  by  other  workers. 
He  also  states  that  when  the  virus  is  injected  intraperitoneally  it 
is  soon  taken  up  by  leucocytes  and  is  not  absorbed  into  the  body 
fluids. 


APPENDIX   B. 

HYDROPHOBIA. 

SYNONYMS. — RABIES  :    FRENCH,  LA  EAGE  :    GERMAN,  LYSSA, 
DIE    HUNDWUTH,    DIE    TOLLWUTH. 

Introductory. — Hydrophobia  is  an  infectious  disease  which  in 
nature  occurs  epidemically  chiefly  among  the  carnivora,  especi- 
ally in  the  dog  and  the  wolf.  Infection  is  carried  by  the  bite 
<>t'  a  rabid  animal  or  by  a  wound  or  abrasion  being  licked  by 
such.  The  disease  can  be  transferred  to  other  species,  and 
\vhrn  once  started  can  be  spread  from  individual  to  individual 
by  the  same  paths  of  infection.  Thus  it  occurs  epidemically 
from  time  to  time  in  cattle,  sheep,  horses,  and  deer,  and  can  be 
communicated  to  man.  It  is  questionable  whether  infection 
can  take  place  from  man  to  man,  as  the  saliva  of  a  person 
suffering  from  hydrophobia  appears  not  to  contain  the  virus. 
It  is  to  be  noted  that  the  virus  is  apparently  extremely  potent, 
a-  cases  of  infection  taking  place  through  an  unabraded  mucous 
membrane  by  the  licking  of  a  rabid  animal  are  on  record,  and  the 
experimental  applications  of  the  virus  to  such  surfaces  as  the 
mucous  membrane  of  the  nose  or  the  conjunctiva  is  often  followed 
by  infection. 

In  Western  Europe  the  disease  is  most  frequently  observed 
in  the  dog;  but  in  Eastern  Europe,  especially  in  Russia, 
epidemics  among  wolves  constitute  a  serious  danger  both  to 
other  animals  and  to  man.  All  the  manifestations  of  the  disease 
point  to  a  serious  affection  of  the  nervous  system  :  but  inasmuch 
as  symptoms  of  excitement  or  of  depression  may  predominate, 
it  is  customary  to  describe  clinically  two  varieties  of  rabies, — (1) 
rabies  proper,  or  furious  rabies  (la  raye  vraie,  la  rar/e  furieuse : 
ill',  ratk  a' I-  \\'utk) ',  and  (2)  dumb  madness  or  paralytic  rabies  (la 
f">/>'  mue :  die  stille  Wuth).  The  disease,  however,  is  essentially 
the  same  in  both  cases.  In  the  dog  the  furious  form  is  the 
more  common.  After  a  period  of  incubation  of  from  three  to 

573 


574  HYDROPHOBIA 

six  weeks,  the  first  symptom  noticed  is  a  change  in  the  animal's 
aspect ;  it  becomes  restless,  it  snaps  at  anything  which  it  touches, 
and  tears  up  and  swallows  unwonted  objects ;  it  has  a  peculiar 
high-toned  bark.  Spasms  of  the  throat  muscles  come  on, 
especially  in  swallowing,  and  there  is  abundant  secretion  of 
saliva ;  its  supposed  special  fear  of  water  is,  however,  a  myth, 
— it  fears  to  swallow  at  all.  Gradually  convulsions,  paralysis, 
and  coma  come  on ;  and  death  supervenes  usually  about  five  days 
after  the  appearance  of  symptoms.  In  the  paralytic  form,  the 
early  symptoms  are  the  same,  but  paralysis  appears  sooner. 
The  lower  jaw  of  the  animal  drops,  from  implication  of  the 
elevator  muscles,  all  the  muscles  of  the  body  become  more  or  less 
weakened,  and  death  ensues  without  any  very  marked  irritative 
symptoms. 

In  man  the  incubation  period  after  infection  varies  from 
fifteen  days  to  seven  or  eight  months,  or  even  longer,  but  is 
usually  about  forty  days.  When  symptoms  of  rabies  are  about 
to  appear,  certain  prodromata,  such  as  pains  in  the  wound  and 
along  the  nerves  of  the  limb  in  which  the  wound  has  been 
received,  may  be  observed.  To  this  succeeds  a  stage  of  nervous 
irritability,  during  which  all  the  reflexes  are  augmented — the 
victim  starting  at  the  slightest  sound,  for  example.  There  are 
spasms,  especially  of  the  muscles  of  deglutition  and  respiration, 
and  cortical  excitement  evidenced  by  delirium  may  occur.  On 
this  follows  a  period  in  which  all  the  reflexes  are  diminished, 
weakness  and  paralysis  are  observed,  convulsions  occur,  and 
finally  coma  and  death  supervene.  The  duration  of  the  acute 
illness  is  usually  from  four  to  eight  days,  and  death  invariably 
results.  The  existence  of  paralytic  rabies  in  man  has  been 
denied  by  some,  but  it  undoubtedly  occurs.  This  is  usually 
manifested  by  paralysis  of  the  limb  in  which  the  infection  has 
been  received,  and  of  the  neighbouring  parts ;  but  while  in  such 
cases  this  is  often  the  first  symptom  observed,  during  the  whole 
of  the  illness  the  occurrence  of  widespread  and  progressive 
paralysis  is  the  outstanding  feature.  In  man  there  also  occur 
cases  where  the  cerebellum  and  also  the  sympathetic  system  seem 
to  be  specially  affected. 

The  Pathology  of  'Hydrophobia. — In  hydrophobia  as  in 
tetanus,  to  which  it  bears  more  than  a  superficial  resemblance, 
the  appearances  discoverable  by  an  ordinary  examination  of  the 
nervous  system,  to  which  all  symptoms  are  naturally  referred,  are 
comparatively  unimportant.  On  naked-eye  examination,  conges- 
tions, and,  it  may  be,  minute  haemorrhages  are  the  only  features 
noticeable.  Microscopically,  leucocytic  exudation  into  the  peri- 


PATHOLOGY  OF  HYDROPHOBIA  575 

vascular  lymphatic  spaces  in  the  nerve  centres  has  been  observed, 
and  in  the  cells  of  the  anterior  cornua  of  the  grey  matter  in  the 
spinal  cord,  and  also  in  the  nuclei  of  the  cranial  nerves,  various 
degenerations  have  been  described.  Round  the  nerve  cells  in 
the  grey  matter  of  the  cord  and  medulla  Babes  described 
accumulations  of  newly-formed  cells,  and  Van  Gehuchten  observed 
a  phagocytosis  of  the  cells  in  the  posterior  root  ganglia  and  also 
in  the  sympathetic  ganglia.  Both  of  these  conditions  were  at  one 
time  thought  to  be  specific  of  rabies,  but  this  has  been  found  not 
to  be  the  case.  In  the  white  matter,  especially  in  the  posterior 
columns,  swelling  of  the  axis  cylinders  and  breaking  up  of  the 
myeline  sheaths  have  been  noted,  and  similar  changes  occur  also 
in  the  spinal  nerves,  especially  of  the  part  of  the  body  through 
which  infection  has  come.  In  the  nervous  system  also  some 
have  seen  minute  bodies  which  they  have  considered  to  be  cocci, 
but  there  is  no  evidence  that  they  are  really  of  this  nature.  The 
changes  in  the  other  parts  of  the  body  are  unimportant. 

Experimental  pathology  confirms  the  view  that  the  nervous 
system  is  the  centre  of  the  disease  by  finding  in  it  a  special 
concentration  of  what,  from  want  of  a  more  exact  term,  we  must 
call  the  hydrophobic  virus.  Pasteur's  first  contribution  to  the 
subject  was  to  show  that  the  most  certain  method  of  infection 
was  by  inserting  the  infective  matter  beneath  the  dura  mater. 
He  found  that  in  the  case  of  any  animal  or  man  dead  of  the 
disease,  injection,  by  this  method,  of  emulsions  of  any  part 
of  the  central  nervous  system,  of  the  cerebro-spinal  fluid,  or 
of  the  saliva,  invariably  gave  rise  to  rabies,  and  also  that  the 
natural  period  of  incubation  was  shortened.  Further,  the 
identity  of  the  furious  and  paralytic  forms  was  proved,  as 
sometimes  the  one,  sometimes  the  other,  was  produced,  what- 
ever form  had  been  present  in  the  original  case.  Inoculation 
into  the  anterior  chamber  of  the  eye  is  nearly  as  efficacious  as 
subdural  infection.  Infection  with  the  blood  or  solid  organs 
of  rabid  animals  does  not  reproduce  the  disease.  There  is 
evidence,  however,  that  the  poison  also  exists  in  such  glands  as 
the  pancreas  and  mamma.  Subcutaneous  infection  with  part  of 
the  nervous  system  of  an  animal  dead  of  rabies  may  or  may  not 
give  rise  to  the  disease. 

In  consequence  of  the  introduction  of  such  reliable  inoculation 
methods,  furthrr  information  has  Urn  acquired  regarding  the 
spread  and  distribution  of  the  virus  in  the  body.  Gaining 
entrance  by  the  infected  wound,  it  early  manifests  its  affinity  for 
the  nervous  tissues.  It  reaches  the  central  nervous  system 
chiefly  by  spreading  up  the  peripheral  nerves.  This  can  be 


576  HYDROPHOBIA 

shown  by  inoculating  an  animal  subcutaneously  in  one  of  its 
limbs,  with  virulent  material.  If  now  the  animal  be  killed 
before  symptoms  have  manifested  themselves,  rabies  can  be 
produced  by  subdural  inoculation  from  the  nerves  of  the  limb 
which  was  infected.  Further,  rabies  can  often  be  produced  from 
such  a  case  by  subdural  infection  with  the  part  of  the  spinal 
cord  into  which  these  nerves  pass,  while  the  other  parts  of  the 
animal's  nervous  system  do  not  give  rise  to  the  disease.  This 
explains  how  the  initial  symptoms  of  the  disease  (pains  along 
nerves,  paralysis,  etc.)  so  often  appear  in  the  infected  part  of  the 
body,  and  it  probably  also  explains  the  fact  that  bites  in  such 
richly  nervous  parts  as  the  face  and  head  are  much  more  likely 
to  be  followed  by  hydrophobia  than  bites  in  other  parts  of  the 
body.  Again,  injection  into  a  peripheral  nerve,  such  as  the 
sciatic,  is  almost  as  certain  a  method  of  infection  as  injection 
into  the  subdural  space,  and  gives  rise  to  the  same  type  of 
symptoms  as  injection  into  the  corresponding  limb.  Intravenous 
injection  of  the  virus,  on  the  other  hand,  differs  from  the  other 
modes  of  infection  in  that  it  more  frequently  gives  rise  to 
paralytic  rabies.  This  fact  Pasteur  explained  by  supposing 
that  the  whole  of  the  nervous  system  in  such  a  case  becomes 
simultaneously  affected.  In  certain  animals  the  virus  seems  to 
have  an  elective  affinity  for  the  salivary  glands,  as  well  as  for 
the  nervous  system.  Roux  and  Nocard  found  that  the  saliva  of 
the  dog  became  virulent  three  days  before  the  first  appearance 
of  symptoms  of  the  disease. 

The  Virus  of  Hydrophobia. — While  a  source  of  infection 
undoubtedly  occurs  in  all  cases  of  hydrophobia,  and  can  usually 
be  traced,  all  attempts  to  determine  the  actual  morbific  cause 
have  been  unsatisfactory.  In  this  connection  various  organisms 
(yeasts,  diphtheroid  bacilli)  have  been  described  as  being 
associated  with  the  disease,  but  none  of  these  have  been  shown 
to  possess  the  capacity  of  producing  immunity  against  the 
ordinary  hydrophobic  virus. 

In  1903  Negri  described  certain  bodies  as  occurring  in  the 
nervous  system  in  animals  dying  of  rabies  to  which  much 
attention  has  since  been  devoted,  and  regarding  the  significance 
of  which  opinion  is  still  divided.  As  Negri's  observations  have 
been  generally  confirmed,  and  as  it  is  probable  that  the 
occurrence  of  the  bodies  is  specific  to  the  disease,  and  that  their 
recognition  is  of  value  for  diagnosis,  we  shall  describe  the 
methods  for  their  demonstration. 

The  method  of  "Williams  and  Lowden  is  to  take  a  piece  of  the  brain 
tissue,  to  squeeze  it  between  a  slide  and  cover-glass,  and,  sliding  off  the 


PATHOLOGY  OF  HYDROPHOBIA      577 

latter,  to  make  a  smear  which  is  then  fixed  in  methyl  alcohol  for  five 
minutes  and  stained  by  Giemsa's  stain  (p.  115)  for  half  an  hour  to  three 
hours  ;  the  preparation  is  then  washed  in  tap  water  for  2-3  min.  and 
dried.  For  rapid  work,  after  fixation,  equal  parts  of  distilled  water  and 
stain  are  used  instead  of  the  more  dilute  mixture. 

For  sections  the  tissues  are  left  in  Zenker's  fluid  l  for  3-4  hours,  then 
placed  in  tap  water  for  five  minutes,  80  per  cent,  alcohol  with  enough 
i i ii line  added  to  give  it  a  port  wine  colour  for  24  hours  ;  95  per  cent, 
alcohol  and  iodine,  24  hours  ;  absolute  alcohol,  4-6  hours  ;  cleared  with 
cedar  oil  and  embedded  in  paraffin  of  melting  point  52°  C.  ;  sections  should 
1»"  •">  to  6  /x  thick.  For  staining,  Mallory's  methylene-blue  eosin  is 
recommended  ;  the  steps  are  as  follows  :  xylol  ;  absolute  alcohol  ;  95 
I >i  r  cent,  alcohol  and  iodine,  J  hour  ;  95  per  cent,  alcohol,  ^  hour  ; 
a  I  isolate  alcohol,  £  hour  ;  eosin  solution  (5-10  per  cent,  aqueous  solution), 
20  minutes  ;  rinse  in  tap  water  ;  Unna's  polychrome  methylene-blue 
solution  diluted  1-4  with  distilled  water,  15  minutes  ;  differentiation  in 
!»5  per  cent,  alcohol  for  1-5  minutes  (the  preparation  being  kept  in 
motion  and  its  progress  watched  with  a  low  power)  ;  rapid  and  careful 
dehydration  and  clearing. 

Frothingham  recommends  a  method  of  making  "impression  prepara- 
tions "  of  the  brain.  The  part  (e.g.  hippocampus)  is  laid  on  a  piece  of 
wood  whose  porosity  causes  it  to  adhere  ;  a  clean  slide  is  then  lowered 
upon  the  tissue  and  slight  pressure  applied  ;  on  raising  the  slide  a 
thin  film  of  cells  preserving  their  original  arrangement  is  lifted  off, 
and  this  can  be  fixed  and  stained  like  a  smear,  van  Gieson's  method 
being  used  by  this  author. 

The  Xegri  bodies  (Plate  IV.,  Fig.  16)2  vary  much  in  size, 
m'Msuring  from  *5  to  25  //,.  They  are  round,  oval,  or  angular 
in  outline.  They  are  found  in  the  protoplasm  of  the  nerve  cells 
and  of  their  processes.  When  examined  in  unstained  prepara- 
tions,  they  are  seen  to  have  a  sharply  defined  outline,  and  some 
of  the  features  of  the  internal  structure  presently  to  be  described 
can  be  noted.  With  regard  to  staining  reactions,  they  are 
!  rankly  eosinophil  for  certain  combinations  containing  eosin, 
e.y.  alcoholic  eosin-methylene-blue,  Mann's  eosin  mixture,  and,  in 
certain  circumstances,  Leishman's  stain.  For  the  finer  differen- 
tiation of  the  internal  structure,  Negri  employed  Giemsa's  stain. 
With  this  stain  and  under  high  magnification  the  groundwork 
of  the  body  is  a  pale  blue ;  in  it  there  appear  certain  round  or 
oval,  multiple  or  single  formations,  of  varying  size,  stained  pink, 
sometimes  occupying  nearly  the  whole  of  the  body,  sometimes 
being  relatively  small  (grosse  Innenformationen).  In  addition, 

1  Zenker's  fluid  is  of  the  following  composition  :  potassium  bichromate 
•J-f>  .m-.,  sodium  sulphate  1  gr.,  perchloride  of  mercury  5  gr.,  glacial  acetic 
a>-i'l  5  c.c.,  water  to  100  c.c.  Dissolve  the  perchloride  of  mercury  and  the 
l.ulirouiate  of  potassium  in  the  water  with  the  aid  of  heat  and  add  the 
a.-rtir  acid. 

-  For  the  material  from  which  this  preparation  was  made  we  are  indebted  to 
nipt.  W.  F.  Harvey,  I. M.S. 

37 


578  '  HYDROPHOBIA 

both  inside  the  larger  formations  and  in  the  general  protoplasm 
of  the  body  are  smaller  red  or  violet-red  granules,  occurring 
singly  or  in  clumps  (kleine  Innenformationen).  With  the  eosin 
dyes  named  above,  and  magnifications  of  800  to  1000,  the 
smaller  bodies  appear  a  homogeneous  reddish  pink,  and  in 
the  larger  bodies  the  outlines  of  the  larger  internal  formations 
can  be  recognised  (see  Plate).  With  Mallory's  stain  they  present 
similar  appearances  with  a  bluish  stippling  of  the  protoplasm. 

The  Negri  bodies  have  been  found  in  practically  98  per  cent, 
of  cases  of  street  rabies  examined  by  many  observers  in  different 
parts  of  the  world.  Numerous  control  observations  on  other 
toxic  conditions  of  the  nervous  system,  especially  where  these 
are  characterised  by  spasms,  have  been  made,  and  although 
occasionally,  e.g.  in  tetanus,  a  somewhat  similar  appearance  has 
been  seen,  at  present  the  consensus  of  opinion  is  in  favour  of  an 
experienced  observer  being  able  to  recognise  the  Negri  bodies  as 
a  specific  appearance  in  nerve  cells.  The  bodies  occur  in  all 
parts  of  the  nervous  system,  but  are  most  common  in  the 
Purkinje  cells  of  the  cerebellum,  and  especially  in  the  cells  of 
the  cornu  Ammonis  (hippocampus  major).  It  is  in  the  last 
situation,  therefore,  that  they  are  generally  looked  for.  They 
are  apparently  not  so  readily  found,  and  may  be  altogether 
absent,  in  animals  dying  from  inoculation  with  the  exalted  fixed 
virus.-  Hitherto  they  have  not  been  found  in  the  salivary 
glands  or  saliva  of  a  rabid  animal. 

While  there  is  a  general  tendency  to  recognise  the  Negri 
bodies  as  being  specific  to  rabies,  great  difference  of  opinion 
exists  as  to  their  true  nature  and  as  to  their  possessing  any 
etiological  significance  in  the  disease.  Negri  himself  looks  upon 
them  as  protozoa,  and  the  organism  has  been  named  by  Calkins 
neuroryctes  hydrophobia*.  The  chief  arguments  advanced  in 
favour  of  this  position  have  been  the  constancy  of  the  occurrence 
of  the  bodies  in  the  brains  of  animals  suffering  from  the  natural 
disease,  and  their  peculiar  structure  which,  such  authorities  as 
Golgi  state,  does  not  correspond  to  any  cellular  degeneration. 
Against  their  protozoal  nature  has  been  urged  their  absence 
from  the  virulent  brains  of  animals  dying  from  fixed  virus,  their 
non-discovery  in  the  infected  saliva,  and  the  fact  that  the  virus 
can  pass  through  a  coarse  filter.  These  objections  have  been  met 
with  the  argument  that  the  smaller  internal  formations  may  be 
the  infective  agent  in  its  essential  form,  and  a  modification  of 
this  view  is  that  the  Negri  body  is  a  cellular  reaction  against 
an  invasion  with  these  ultimate  forms.  The  whole  question 
must  be  looked  upon  as  sub  judice. 


PROPHYLACTIC  TREATMENT  OF  HYDROPHOBIA    579 

There  is  no  doubt  that  between  rabies  and  the  bacterial 
disease's  \\v  have  studied  there  are  at  every  point  analogies,  the 
most  striking  U'ing  the  protective  inoculation  methods,  the  <li- 
co\ cry  of  which  constitutes  the  great  work  of  Pasteur ;  and  every- 
thing points  to  a  micro-organism  being  the  cause.  The  organism, 
whatever  it  is,  is,  in  its  infective  form,  probably  very  small,  as  it 
can  pass  through  the  coarser  Berkefeld  filters,  and  also  occasion- 
ally through  the  coarser  Chamberland  candles.  Evidence  that  it 
is  the  organism  itself  which  passes  through,  is  found  in  the  fact 
that  when  iin  animal  dies  from  infection  with  the  filtrate,  a 
small  portion  of  its  central  nervous  system  will  originate  the 
disease  in  a  fresh  animal.  Judging  from  our  knowledge  of 
similar  diseases,  we  would  strongly  suspect  that  it  is  actually 
present  in  a  living  condition  in  the  central  nervous  system,  the 
>ali\a.  etc.,  which  yield  what  we  have  called  the  hydrophobic 
virus,  for  by  no  mere  toxin  could  the  disease  be  transmitted 
through  a  series  of  animals,  as  we  shall  presently  see  can  be 
done.  A  toxin  may,  however,  be  concerned  in  the  production 
of  the  pathogenic  effects.  Reinlinger  found  that  death  with 
paralytic  symptoms  followed  the  injection  of  filtered  virus,  but 
that  the  nervous  system  of  the  dead  animals  sometimes  did  not 
reproduce  rabies.  He  explains  this  occurrence  by  supposing  that 
the  filtrate  contained  a  toxin  but  not  the  actual  infective  agent. 
The  resistance  of  the  virus  to  external  agents  varies.  Thus  a 
iMTvous  system  containing  it  is  virulent  till  destroyed  by  putre- 
faction ;  it  can  resist  the  prolonged  application  of  a  temperature 
of  from  -  10°  to  —  20°  C.,  but,  on  the  other  hand,  it  is  rendered 
non-virulent  by  one  hour's  exposure  at  50°  C.  Again,  its 
potency  probably  varies  in  nature  according  to  the  source. 
Thus,  while  the  death-rate  among  persons  bitten  by  mad  dogs  is 
about  16  per  cent.,  the  corresponding  death-rate  after  the  bites 
ot  wolves  is  80  per  cent.  Here,  however,  it  must  be  kept  in 
view  that,  as  the  wolf  is  naturally  the  more  savage  animal,  the 
number  and  extent  of  the  bites,  i.e.  the  number  of  channels  of 
entrance  of  the  virus  into  the  body  and  the  total  dose,  are 
urreater  than  in  the  case  of  persons  bitten  by  dogs.  As  we  shall 
see,  alterations  in  the  potency  of  the  virus  can  certainly  be 
effected  by  artificial  means. 

The  Prophylactic  Treatment  of  Hydrophobia. — Until  the 
publication  of  Pasteur's  researches  in  1885,  the  only  means 
adopted  to  prevent  the  development  of  hydrophobia  in  a  person 
bitten  by  a  rabid  animal  had  consisted  in  the  cauterisation  of 
the  wound.  Such  a  procedure  was  undoubtedly  not  without 
effect.  It  has  been  shown  that  cauterisation  within  five  minutes 


580  HYDROPHOBIA 

of  the  infliction  of  a  rabic  wound  prevents  the  disease  from 
developing,  and  that  if  done  within  half  an  hour  it  saves  a 
proportion  of  the  cases.  After  this  time,  cauterisation  only 
lengthens  the  period  of  incubation ;  but,  as  we  shall  see 
presently,  this  is  an  extremely  important  effect. 

The  work  of  Pasteur  has,  however,  revolutionised  the  whole 
treatment  of  wounds  inflicted  by  hydrophobic  animals.  Pasteur 
started  with  the  idea  that,  since  the  period  of  incubation  in  the 
case  of  animals  infected  subdurally  from  the  nervous  systems  of 
mad  dogs  is  constant  in  the  dog,  the  virus  has  been  from  time 
immemorial  of  constant  strength.  Such  a  virus,  of  what  might 
be  called  natural  strength,  is  usually  referred  to  in  his  works  as 
the  virus  of  la  rage  des  rues,1  in  the  writings  of  German  authors 
as  the  virus  of  die  Strasswuth.  Pasteur  found  on  inoculating 
a  monkey  subdurally  with  such  a  virus,  and  then  inoculating 
a  second  monkey  from  the  first,  and  so  on  with  a  series  of 
monkeys,  that  it  gradually  lost  its  virulence,  as  evidenced  by 
lengthened  periods  of  incubation  on  subdural  inoculation  of 
dogs,  until  it  wholly  lost  the  power  of  producing  rabies  in  dogs, 
when  introduced  subcutaneously.  When  this  point  had  been 
attained,  its  virulence  was  not  diminished  by  further  passage 
through  the  monkey.  On  the  other  hand,  if  the  virus  of  la 
rage  des  rues  were  similarly  passed  through  a  series  of  rabbits 
or  guinea-pigs,  its  virulence  was  increased  till  a  constant  strength 
(the  virus  fixe)  was  attained, — constancy  of  strength  being  in- 
dicated by  the  unvarying  recurrence  of  paresis  on  the  sixth  day. 
Pasteur  had  thus  at  command  three  varieties  of  virus — that  of 
natural  strength,  that  which  had  been  attenuated,  and  that 
which  had  been  exalted.  He  further  found  that,  commencing 
with  the  subcutaneous  injection  of  a  weak  virus,  and  following 
this  up  with  the  injection  of  the  stronger  varieties,  he  could 
ultimately,  in  a  very  short  time,  immunise  dogs  against  subdural 
infection  with  a  virus  which,  under  ordinary  conditions,  would 
certainly  have  caused  a  fatal  result.  He  also  elucidated  the 
fact  that  the  exalted  virus  contained  in  the  spinal  cords  of 
rabbits  such  as  those  referred  to,  could  be  attenuated  so  as  no 
longer  to  produce  rabies  in  dogs  by  subcutaneous  injection. 
This  was  done  by  drying  the  cords  in  air  over  caustic  potash  (to 

1  While  Pasteur's  original  statements  regarding  the  constancy  of  the 
virulence  of  the  street-virus  were  probably  accurate  for  the  street  dogs  of 
Paris,  it  has  been  found  that  if  the  general  virulence  of  virus  derived  from 
animals  in  nature  be  studied,  considerable  variation  occurs.  It  is  now 
usual  to  apply  the  term  street- virus  to  any  virus  derived  from  an  animal 
becoming  rabid  under  natural  conditions  of  infection. 


PROPHYLACTIC  TREATMENT  OF  HYDROPHOBIA    581 

absorb  the  moisture),  the  diminution  of  virulence  being  propor- 
tional to  the  length  of  time  during  which  the  cords  were  kept. 
Accordingly,  by  taking  a  series  of  such  spinal  cords  kept  for 
various  periods  of  time,  he  was  supplied  with  a  series  of  vaccines 
of  different  strengths.  Pasteur  at  once  applied  himself  to  find 
whether  the  comparatively  long  period  of  incubation  in  man  could 
not  be  taken  advantage  of  to  "  vaccinate "  him  against  the 
disease  before  its  gravest  manifestation  took  place.  The 
following  is  the  record  of  the  first  case  thus  treated.  The  technique 
was  to  rub  up  in  a  little  sterile  bouillon  a  small  piece  of  the 
oord  used,  and  inject  it  under  the  skin  by  means  of  a  hypodermic 
syringe.  The  first  injection  was  made  with  a  very  attenuated 
virus,  i.e.  a  cord  fourteen  days  old.  In  subsequent  injections 
the  strength  of  the  virus  was  gradually  increased,  as  shown  in 
the  table  :— 

July    7,  1885,  J)  A.M.,  cord  of  June  23,  i.e.  14  days  old. 


7 

6  P.M. 

25 

12 

8 

9  A.M. 

27 

11 

8 

6  P.M. 

29 

9 

9 

11  A.M.,  cord  o 

July  1 

8 

10 

3 

7 

11 

5 

G 

U 

7 

5 

13 

9 

4 

14 

11 

3 

15 

13 

2 

16 

15 

1  day  old. 

The  patient  never  manifested  the  slightest  symptom  of  hydro- 
phobia. Other  similarly  favourable  results  followed ;  and  this 
prophylactic  treatment  of  the  disease  quickly  gained  the  con- 
fidence of  the  scientific  world,  which  it  still  retains. 

An  important  modification  in  the  method  which  further  experience  led 
Pasteur  to  make  was  in  the  treatment  of  serious  cases,  such  as  multiple 
bites  from  wolves,  extensive  bites  about  the  head,  especially  in  children, 
cases  which  come  under  treatment  at  a  late  period  of  the  incuhation 
stage,  and  cases  where  the  wounds  have  not  cicatrised.  In  such  cases 
the  stages  of  the  treatment  are  condensed.  Thus  on  the  first  day,  say 
at  11  A.M.  and  4  P.M.  and  9  P.M.,  cords  of  12,  10,  and  8  days  respectively 
are  used  ;  on  the  second  day,  cords  of  6,  4,  and  2  days ;  on  the  third 
•lay,  cords  of  1  day  ;  on  the  fourth  day,  cords  of  8,  6,  and  4  days ;  on 
the  fifth,  cords  of  3  and  2  days  ;  on  the  sixth,  cords  of  1  day  ;  and  so  on 
for  ten  days.  In  each  case  the  average  dose  is  about  2  c.c.  of  the  emulsion. 

The  details  of  the  prophylactic  treatment  with  regard  to 
dosage  and  virulence  of  material  used  vary  in  different  Pasteur 
institutes.  The  most  important  modification  which  has  within 


582  HYDROPHOBIA 

recent  times  taken  place  is  the  substitution  by  Hogyes  of 
increasing  concentrations  of  a  fairly  fresh  virulent  rabbit's 
cord  for  emulsions  of  cords  subjected  to  decreasing  periods  of 
drying.  Equally  good  results  apparently  are  obtained  by  this 
method,  and  it  is  stated  that  in  cases  so  treated  certain 
symptoms  sometimes  following  the  ordinary  treatment,  the 
gravest  of  which  may  be  the  occurrence  of  temporary  paralyses, 
are  not  so  frequently  observed.  This,  according  to  Harvey  and 
McKendrick,  who  have  studied  the  subject  very  fully,  may  be 
due  to  the  fact  that  a  smaller  amount  of  nerve  tissue  is  injected 
under  the  Hogyes  system. 

The  success  of  the  treatment  has  been  very  marked.  The  statistics  of 
the  cases  treated  in  Paris  are  published  quarterly  in  the  Amiales  de 
I'lnstitut  Pasteur,  and  general  summaries  of  the  results  of  each  year  are 
also  prepared.  As  we  have  said,  the  ordinary  mortality  formerly  was 
16  per  cent,  of  all  persons  bitten.  During  the  ten  years  1886-95, 
17,337  cases  were  treated,  with  a  mortality  of  '48  per  cent.  It  has  been 
alleged  that  many  people  arc  treated  who  have  been  bitten  by  dogs  that 
were  not  mad.  This,  however,  is  not  more  true  of  the  cases  treated  by 
Pasteur's  method  than  it  was  of  those  on  which  the  ordinary  mortality 
of  16  per  cent,  was  based,  and  care  is  taken  in  making  up  the  statistics 
to  distinguish  the  cases  into  three  classes.  Class  A  includes  only  persons 
bitten  by  dogs  proved  to  have  had  rabies,  by  inoculation  in  healthy 
animals  of  parts  of  the  central  nervous  system  of  the  diseased  animal. 
Class  B  includes  those  bitten  by  dogs  that  a  competent  veterinary  surgeon 
has  pronounced  to  be  mad.  Class  C  includes  all  other  cases.  During 
1895,  122  cases  belonging  to  Class  A  were  treated,  with  no  deaths  ;  940 
belonging  to  Class  B,  with  two  deaths  ;  and  449  belonging  to  Class  C, 
with  no  deaths.  Besides  the  Institute  in  Paris,  similar  institutions  exist 
in  other  parts  of  France,  in  Italy,  and  especially  in  Russia,  as  well  as 
in  other  parts  of  the  world  ;  and  in  these  similar  success  has  been 
experienced.  It  may  be  now  taken  as  established,  that  a  very  grave 
responsibility  rests  on  those  concerned,  if  a  person  bitten  by  a  mad 
animal  is  not  subjected  to  the  Pasteur  treatment.  Sometimes  during  or 
after  treatment  there  appear  slight  paralytic  symptoms  with  obstinate 
constipation  and  it  may  be  retention  of  urine,  but  these  usually  pass  off 
within  a  few  weeks  and  leave  behind  no  ill  effects. 

The  principles  underlying  the  prophylactic  treatment  of 
rabies  raise  questions  of  the  highest  interest  from  the  standpoint 
of  immunity.  The  prime  fact  is,  as  has  been  stated,  the  taking 
advantage  of  the  long  period  of  incubation  of  the  disease  in  man 
to  neutralise  an  infection  which  may  be  supposed  to  be  gradually 
gathering  force.  We  have  here  again  to  deal  with  an  example 
of  the  reinforcement  of  the  natural  powers  of  resistance  of  the 
body  in  order  to  enable  it  to  cope  with  a  local  pathological 
change,  the  locus  in  this  case  being  the  nervous  system.  We 
are  thus  unable  at  present  to  give  a  rational  explanation  of  the 


MKTHODS  5S3 

ellicacy  of  the  treatment,  but  again  attention  may  be  directed  to 
the  bearing  which  the  development  of  hypersensitiveness  may 
have  to  the  occurrence  of  the  phenomena  of  infective  disease, 
and  Harvey  and  McKendrick  draw  attention  to  the  fact  that 
some  of  the  concurrent  symptoms  associated  with  the  treatment 
closely  resemble  anapliylactic  phenomena. 

J /////•'//,/»•  .s'r  rum. — In  the  early  part  of  the  nineteenth  century 
an  Italian  physician,  Valli,  showed  that  immunity  against  rabies 
could  In-  conferred  by  administering  through  the  stomach  pro- 
gressively increasing  doses  of  hydrophobic  virus.  Following  up 
this  observation,  Tizzoni  and  Centanni  have  attenuated  rabic 
virus  by  submitting  it  to  peptic  digestion,  and  have  immunised 
animals  by  injecting  gradually  increasing  strengths  of  such  virus. 
This  method  is  usually  referred  to  as  the  Italian  method  of 
immunisation.  The  latter  workers  showed  from  this  that  the 
serum  of  animals  thus  immunised  could  give  rise  to  passive 
immunity  in  other  animals  ;  and  further,  that  if  injected  into 
animals  from  seven  to  fourteen  days  after  infection  with  the 
virus,  it  prevented  the  latter  from  producing  its  fatal  effects, 
even  when  symptoms  had  begun  to  manifest  themselves.  They 
fiirthur  succeeded  in  producing  in  the  sheep  and  the  dog  an 
immunity  equal  to  from  1-25,000  to  1-50,000  (vide  p.  525),  and 
they  recommended  the  use,  in  severe  cases,  of  the  serum  of  such 
animals  in  addition  to  the  treatment  of  the  patient  by  the 
Pasteur  method.  A  like  serum  has  been  obtained  from  animals 
treated  by  the  ordinary  Pasteur  method. 

Methods. — (1)  Diagnosis. — Recent  work  with  regard  to  the 
specificity  of  the  Negri  bodies  for  rabies  has  led  to  a  modifica- 
tion in  the  procedure  to  be  adopted.  Formerly  it  was  advisable 
if  possible  to  keep  an  animal  suspected  of  rabies  alive  for  the 
observation  of  symptoms.  While  the  clinical  history  of  the 
animal  ought  to  be  carefully  obtained,  greater  information  will 
be  obtained  by  examination  of  its  hippocampus.  The  animal 
should  therefore  l»e  killed  and  the  brain  removed  after  reflecting 
the  scalp  and  cutting  through  the  calvarium  with  a  sharp  chisel. 
The  brain  is  laid  down,  vertex  uppermost,  and  the  upper  parts 
of  one  hemisphere  are  removed  in  thin  horizontal  slices  till  the 
anterior  part  of  the  lateral  ventricle  is  reached.  The  roof  of  the 
ventricle  is  then  cut  away  with  a  probe-pointed  bistoury,  and 
the  hippocampus  will  be  recognised  as  the  laterally  arched  ridge 
which  forms  the  floor  of  the  ventricle.  This  may  be  transversely 
incised  and  parts  removed  for  the  making  of  smears  and  sections 
(p.  r,76).  ^ 

In  addition  to  microscopic  examination,  a  small  piece  of  the 


584  HYDROPHOBIA 

medulla  or  cord  of  the  suspected  animal  must  be  taken,  with  all 
aseptic  precautions,  rubbed  up  in  a  little  sterile  '75  per  cent, 
sodium  chloride  solution,  and  injected  by  means  of  a  syringe 
beneath  the  dura  mater  of  a  rabbit,  the  latter  having  been 
trephined  over  the  cerebrum  by  means  of  the  small  trephine 
which  is  made  for  the  purpose.  In  rabies  in  the  rabbit 
symptoms  of  paresis  usually  occur  in  from  six  to  twenty-three 
days  and  death  in  fifteen  to  twenty-five  days.  When  the  material 
for  inoculation  has  to  be  sent  any  distance,  this  is  best  effected 
by  packing  the  head  of  the  animal  in  ice.  The  virulence  of 
organs  is  not  lost,  however,  if  they  are  simply  placed  in  sterile 
water  or  glycerin  in  well-stoppered  bottles. 

(2)  Treatment. — Every  wound  inflicted  by  a  rabid  animal 
ought  to  be  cauterised  with  the  actual  cautery  as  soon  as 
possible.  By  such  treatment  the  incubation  period  will  at  any 
rate  be  lengthened,  and  therefore  there  will  be  better  opportunity 
for  the  Pasteur  inoculation  method  being  efficacious.  The 
person  ought  then  to  be  sent  to  the  nearest  Pasteur  Institute  for 
treatment.  It  is  of  great  importance  that  in  such  a  case  the 
nervous  system  of  the  animal  should  also  be  sent,  in  order  that 
the  diagnosis  may  be  certainly  verified. 


APPENDIX  C. 

MALARIAL  FEVER. 

IT  has  now  been  conclusively  proved  that  the  cause  of 
malarial  fever  is  a  protozoon  of  which  there  are  several  species. 
They  belong  to  the  haemosporidia  (a  sub-class  of  the  sporozoa) 
which  are  blood  parasites,  infecting  the  red  corpuscles  of  mam- 
mals, reptiles,  and  birds.  The  parasite  was  formerly  known  as 
the  licp.inatozoon  or  plasmodium  malarice,  although  the  use  of  the 
latter  form  is  incorrect ;  the  term  hcemamoeba  is,  however,  now 
generally  employed.  The  parasite  was  first  observed  by  Laveran 
in  1880,  and  his  discovery  received  confirmation  from  the 
i  in  k'i»endent  researches  of  Marchiafava  and  Celli,  and  later  from  the 
researches  of  many  others  in  various  parts  of  the  world.  Golgi 
supplied  valuable  additional  information,  especially  in  relation 
t<>  the  .sporulation  of  the  organism  and  the  varieties  in  different 
types  of  malarial  fever.  In  this  country  valuable  work  on  the 
subject  was  done  by  Mauson,  and  to  him  specially  belongs  the 
credit  of  regarding  the  exfiagellation  of  the  organism  as  a 
preparation  for  an  extra-corporeal  phase  of  existence.  By 
induction  he  arrived  at  the  belief  that  the  cycle  of  existence 
outside  the  human  body  probably  took  place  in  the  mosquito. 
It  was  specially  in  order  to  discover,  if  possible,  the  parasite  in 
this  insect,  that  Ross  commenced  his  long  series  of  observations, 
which  were  ultimately  crowned  with  success.  After  patient  and 
persistent  search,  he  found  rounded  pigmented  bodies  in  the  wall 
of  the  stomach  of  a  dapple-winged  mosquito  (a  species  of 
Anopheles)  which  had  been  fed  on  the  blood  of  a  malarial 
patient.  The  pigment  in  these  bodies  was  exactly  similar  to 
that  in  the  malarial  parasite,  and  he  excluded  the  possibility  of 
their  representing  anything  else  than  a  stage  in  the  life  cycle  of 
the  organism.  He  confirmed  this  discovery  and  obtained  cor- 
responding results  in  the  case  of  the  proteosoma  infection  of 
birds,  where  the  parasite  is  closely  related  to  that  of  malaria. 
In  birds  affected  with  this  organism,  he  was  able  to  trace  all 


586  MALAEIAL  FEVER 

stages  of  its  development,  from  the  time  it  entered  the  stomach 
along  with  the  blood,  till  the  time  when  it  settled  'in  a  special 
form  in  the  salivary  glands  of  the  insect.  Ross's  results  were 
published  in  1898.  Exactly  corresponding  stages  were  after- 
wards found  in  the  case  of  the  different  species  of  the  human 
parasite,  by  Grassi,  Bignami,  and  Bastianelli;  and  these  with 
other  Italian  observers  also  supplied  important  information 
regarding  the  transmission  of  the  disease  by  infected  mosquitoes. 
Abundant  additional  observations,  with  confirmatory  results, 
were  supplied  by  Koch,  Daniels,  Christophers,  Stephens,  and 
others.  Wherever  malaria  has  been  studied  the  result  has  been 
the  same.  Lastly,  we  may  mention  the  striking  experiment 
carried  out  by  Manson  by  means  of  mosquitoes  fed  on  the  blood 
of  patients  in  Italy  suffering  from  mild  tertian  fever.  The 
insects,  after  being  thus  fed,  were  taken  to  London  and  allowed 
to  bite  the  human  'subject,  Manson's  son,  Dr.  P.  Thurburn 
Manson,  offering  himself  for  the  purpose.  The  result  was  that 
infection  occurred;  the  parasites  appeared  in  the  blood,  and 
were  associated  with  an  attack  of  tertian  fever.  Ross's  discovery 
has  not  only  been  a  means  of  elucidating  the  mode  of  infection, 
but,  as  will  be  shown  below,  has  also  supplied  the  means  of 
successfully  combating  the  disease. 

From  the  zoological  point  of  view  the  mosquito  is  regarded 
as  the  definitive  host  of  the  parasite,  the  human  subject  as  the 
intermediate  host.  But  in  describing  the  life  history,  it  will  be 
convenient  to  consider,  first,  the  cycle  in  the  human  body,  and, 
secondly,  that  in  the  mosquito.  Various  terms  have  been 
applied  to  the  various  stages,  but  we  shall  give  those  now 
generally  used. 

The  Cycle  in  the  Human  Subject. — With  regard  to  this 
cycle  (Plate  V.,  Fig.  21  a — /),  it  may  be  stated  that  the  parasite 
is  conveyed  by  the  bite  of  the  mosquito  in  the  form  of  a  small 
filamentous  cell — sporozoite  or  exotospore,  which  penetrates  a  red 
corpuscle  and  becomes  a  small  amoeboid  organism  or  amcebula. 
There  is  then  a  regularly  repeated  asexual  cycle  of  the  parasite 
in  the  blood,  the  length  of  which  cycle  determines  the  type  of 
the  fever.  During  this  cycle  there  is  a  growth  of  the  amoebuhie 
or  trophozoites  within  the  red  corpuscles  up  to  their  complete 
development ;  schizogony  (formerly  called  sporulation)  then 
occurs.  The  onset  of  the  febrile  attack  corresponds  with  the 
stage  of  schizogony  and  the  setting  free  of  the  merozoites  or 
enhaemospores,  i.e.  with  the  production  of  a  fresh  brood  of 
parasites.  These  soon  become  attached  to,  and  penetrate  into, 
the  interior  of  the  red  corpuscles,  becoming  intra-corpuscular 


FORMS  OF  THE  MALARIAL  PARASITE        587 

annul)  u  he ;  the  cycle  is  thus  completed.  The  parasites  are  most 
numerous  in  the  blood  during  the  development  of  the  pyivxia, 
and,  further,  they  are  also  much  more  abundant  in  the  internal 
organs  than  in  .the  peripheral  blood;  in  the  malignant  type,  for 
example,  the  process  of  schizogony  is  practically  confined  to  the 
former, 

In  addition  to  these  forms  which  are  part  of  the  ordinary 
asexual  cycle,  there  are  derived  from  the  anuebuhe  other  forms, 
\\hich  arc  called  i/n/m-fnri/fes,  or  sexual  cells.  These  remain 
unaltered  during  successive  attacks  of  pyrexia,  and  undergo  no 
further  change  until  the  blood  is  removed  from  the  human  body. 
Fn  the  simple  tertian  and  quartan  fevers  (vide  infra)  the  gameto- 
cvtes  resemble  somewhat  in  appearance  the  fully  developed 
aiiKL'huUe  before  sporulation,  whereas  in  the  malignant  type  they 
have  a  characteristic  crescent-like  or  sausage-shaped  form ;  hence 
they  are  often  spoken  of  as  "crescentic  bodies"  (Plate  V.,  Fig. 

--.'.<; .-/). 

The  various  forms  of  the  parasite  seen  in  the  human  blood 
may  now  be  described  more  in  detail. 

I .  '/'//<•  Merozoites  (Enhcemospores,  Lankester)  are  the  youngest 
and  smallest  forms  resulting  from  the  segmentation  of  the 
adult  amoebula  or  schizont.  They  are  of  round  or  oval 
shape  and  of  small  size,  usually  not  exceeding  2  //,  in 
dia meter;  the  size,  however,  varies  somewhat  in  the  different 
t  \pi-s  of  fever.  A  nucleus  and  peripheral  protoplasm  can  be 
distinguished  (Fig.  162).  The  former  appears  as  a  small 
rounded  body  which  usually  remains  unstained,  but  contains  a 
minute  mass  of  chromatin  which  stains  a  deep  red  with  the 
I  (omanowsky  method,  the  peripheral  protoplasm  being  coloured 
fairly  deeply  with  methylene-blue.  The  merozoites  show  little  or 
no  anneltoid  movement;  at  first  free  in  the  plasma,  they  soon 
attack  the  red  corpuscles,  where  they  become  the  intra-corpuscular 
an  in  -Iniho.  If  the  blood,  say  in  a  mild  tertian  case,  be  examined 
in  the  early  stages  of  pyrexia,  one  often  finds  at  the  same  time 
schi/onts,  free  merozoites,  and  the  young  amcebuhe  within  the 
the  red  corpuscles. 

•_' .  / / /  / / •"  -•-,, , -f , uscular  A mcebula'  or  Trophozoites. — These  include 
the  parasites  which  have  attacked  the  red  corpuscles ;  they  are  at 
first  situated  on  the  surface  of  the  latter  but  afterwards  penetrate 
their  substance.  They  usually  occur  singly  in  the  red  corpuscles, 
but  sometimes  two  or  more  may  be  present  together.  As  seen 
in  fresh  blood,  the  youn^-st  "i  smallest  forms  are  minute  colour- 
It  -ss  specks,  of  about  the  same  si/e  as  the  spores;  they  exhibit 
more  or  less  active  amoeboid  movement,  showing  marked 


588  MALARIAL  FEVER 

variations  in  shape.  The  amount  and  character  of  the  amoeboid 
movement  varies  somewhat  in  different  types  of  fever.  As  they 
increase  in  size,  pigment  appears  in  their  interior  as  minute  dark 
brown  or  black  specks,  and  gradually  becomes  more  abundant 
(Figs.  158,  159,  Plate  V.,  Fig.  21  c,  d,  e,  Fig.  22  e).  This 
pigment  is  elaborated  from  the  haemoglobin  of  the  red  cor- 
puscles, the  parasite  growing  at  the  expense  of  the  latter. 
The  red  corpuscles  thus  invaded  may  remain  unaltered  in 
appearance  (quartan  fever),  may  become  swollen  and  pale 
(tertian  fever),  or  somewhat  shrivelled  and  of  darker  tint 
(malignant  fever).  In  stained  specimens  a  nucleus  may  be 
seen  in  the  parasite  as  a  pale  spot  containing  chromatin  which 
may  be  arranged  as  a  single  concentrated  mass  or  as  several 
separated  granules,  the  chromatin  being  coloured  a  deep  red  by 
the  Romanowsky  method.  The  protoplasm  of  the  parasite, 
which  is  coloured  of  varying  depth  of  tint  with  methylene-blue, 
shows  great  variation  in  configuration  (Fig.  159).  The  young 
parasites  not  infrequently  present  a  "ring-form,"  a  portion  of 
the  red  corpuscle  being  often  enclosed  by  the  parasite.  These 
ring-forms  are  met  with  in  all  the  varieties  of  the  parasite,  but 
they  are  especially  common  in  the  case  of  the  malignant  parasite, 
where  they  are  of  smaller  size  and  of  more  symmetrical  form 
than  in  the  others  (Fig.  163). 

Within  the  red  corpuscles  the  parasites  gradually  increase 
in  size  till  the  full  adult  form  is  reached  (Fig.  160).  In  this 
stage  the  parasite  loses  its  amoeboid  movement  more  or  less 
completely,  has  a  somewhat  rounded  form,  and  contains  a 
considerable  amount  of  pigment.  In  the  malignant  form  it  only 
occupies  a  fraction  of  the  red  corpuscle.  The  adult  parasites 
may  then  undergo  schizogony,  but  not  all  of  them  do  so ;  some 
become  degenerated  and  ultimately  break  down. 

3.  Schizonts. — In  the  process  of  schizogony  the  nuclear 
outline  becomes  lost,  and  the  chromatin  becomes  divided  into 
a  number  of  small  granules  which  are  scattered  through  the 
protoplasm ;  the  latter  then  undergoes  corresponding  segmenta- 
tion and  the  small  merozoites  or  enhsemospores  result.  The 
pigment  during  the  process  becomes  aggregated  in  the  centre 
and  is  surrounded  by  a  small  quantity  of  residuary  protoplasm. 
(Schaudinn  has  found  in  the  case  of  the  tertian  parasite  that 
schizogony  begins  by  a  sort  of  primitive  mitosis,  which  is  then 
followed  by  simple  multiple  fission.)  The  merozoites  are  of 
rounded  or  oval  shape,  as  above  described,  and  are  set  free 
by  the  rupture  of  the  envelope  of  the  red  corpuscles.  The 
pigment  also  becomes  free  and  may  be  taken  up  by  leucocytes. 


FIG.  159. 


FIG.  160. 


FIG.  161.  FIG.  162. 

FIGS.  157-162.— Various  phases  of  the  benign  tertian  parasite. 

FIG.  157.   Several  young  ring-shaped  amuebulse  within  the  red  corpuscles,  one  of 

the  latter  enlarged  and  showing  a  dotted  appearance.     Fig.  158.  A  larger  amoebula 

Containing  pigment  granules.     Fig.  159.  Two  large  amoebulae,  exemplifying  the  great 

variation  in  form.    Fig.  160.  Large  amoebula  assuming  the  spherical  form  and  showing 

isolated  fragments  of  chromatin— preparatory  to  schizogony.      Fig.  161.  Schizont, 

which  has  produced  eighteen  merozoites,  each  of  which  contains  a  small  collection 

of  chromatin.     Fig.  162.  A  number  of  merozoites  which  have  just  been  sit  free  m 

the  plasma,     x  1000. 


FIG.  16-3. 


FIG.  164. 


FIG.  167. 


FIG.  168. 


FIGS.  163-168. — Exemplifying  phases  of  the  malignant  parasite. 

FIG.  163.  Two  small  ring-shaped  amoebulae  within  the  red  corpuscles.  Fig.  164.  A 
"crescent"  or  gamete  showing  the  envelope  of  the  red  corpuscles  ;  also  an  amoebula. 
Figs.  165-168  illustrate  the  changes  in  form  undergone  by  the  crescents  outside  the 
body.  In  the  interior  of  the  spherical  form  in  Fig.  167  evidence  of  the  flagella  can  be 
seen.  Fig.  168.  A  male  gametocyte  which  has  undergone  exflagellation,  showing  the 
thread-like  microgametes  or  spermatozoa  attached  at  the  periphery,  x  1000.  (The 
figures  in  this  plate  are  from  preparations  kindly  lent  by  Sir  Patrick  Manson.) 


FOIJMS  OF  THE  MALARIAL  PARASITE         591 

The  number  and  arrangement  of  the  merozoites  within  the 
srhi/ont  vary  in  the  different  types.  In  the  quartan  there  are 
6-12,  and  the  segmentation  is  in  a  radiate  manner,  giving  rise 
to  the  characteristic  daisy  head  appearance;  in  the  tertian  they 
number  ].")  •_'<)  or  more,  and  have  a  somewhat  rosette-like 
arrangement  (Fig.  161);  in  the  malignant  there  are  usually 
6-20  merozoites  of  small  size  and  somewhat  irregularly  arranged. 

Gametocytes. — As  stated  above,  these  are  sexual  cells  which 
are  formed  from  certain  of  the  amoebulce,  and  which  undergo  no 
further  development  in  the  human  subject.  In  the  mild  tertian 
and  quartan  fevers  they  are  rounded  and  resemble  somewhat 
the  largest  amoebulae.  The  female  cells,  macroyametocytes,  are 
of  large  size,  measuring  up  to  1 6  /x  in  diameter ;  they  contain 
coarse  grains  of  pigment,  and  the  protoplasm  stains  somewhat 
deeply  witli  methylene-blue.  The  male  cells,  microffometocytes, 
are  smaller,  and  the  protoplasm  stains  faintly;  the  nucleus, 
umerally  in  the  centre,  is  rich  in  chromatin.  In  the  malignant 
fevers  the  gametocytes  have  the  special  crescentic  or  sausage- 
shaped  form  mentioned  above.  They  measure  8  to  9  /x  in  length, 
and  occasionally  a  fine  curved  line  is  seen  joining  the  extremities 
on  the  concave  aspect,  which  represents  the  envelope  of  the  red 
corpuscle  (Fig.  164).  They  are  colourless  and  transparent,  and 
are  enclosed  by  a  distinct  membrane ;  in  the  central  part  there 
is  a  collection  of  pigment  and  granules  of  chromatin.  The  male 
crescents  can  be  distinguished  from  the  female  by  their  appear- 
ance ;  in  the  former  the  pigment  is  less  dark  and  more  scattered 
through  the  cell,  and  there  are  several  granules  of  chromatin  ; 
in  the  latter  the  pigment  is  dark  and  concentrated,  often  in  a 
small  ring,  and  there  are  one  or  two  masses  of  chromatin  in  the 
centre  of  the  crescent  (Plate  V.,  Fig.  22 /,  g).  According  to  the 
Italian  observers,  the  early  forms  of  the  crescents  are  somewhat 
fusiform  in  shape  and  are  produced  in  the  bone-marrow.  The 
fully  developed  crescents  do  not  appear  in  the  blood  till  several 
days  after  the  onset  of  the  fever,  and  they  may  be  found  a 
considerable  time  after  the  disappearance  of  the  pyrexial  attacks. 
They  are  also  little,  if  at  all,  influenced  by  the  administration 
of  quinine. 

It  is  well  known  that  after  a  patient  has  apparently  recovered 
from  malarial  fever  a  relapse  may  take  place  without  fresh 
infection  occurring,  sometimes  several  years  afterward,  and 
Schaudiim  has  published  interesting  observations  bearing  on 
this  point.  He  has  found,  and  his  observations  on  this  point 
have  been  confirmed,  that  the  macrogametocyte  of  tertian  fever 
may  by  a  process  of  parthenogenesis  give  rise  to  merozoites, 


592  MALARIAL  FEVER 

which  in  their  turn  infect  the  red  corpuscles  and  start  the  cycle 
again.  As  described  and  figured  by  him,  the  chromatin  of  the 
macrogametocyte  divides  first  into  two  portions,  one  of  which  is 
smaller  and  stains  more  deeply  than  the  other.  This  more 
deeply  staining  portion  then  divides,  and  the  protoplasm  becomes 
segmented  as  in  ordinary  schizogony,  and  a  young  brood  of 
parasites  results.  The  more  faintly-staining  chromatin  along 
with  part  of  the  protoplasm  breaks  up  and  disappears. 

The  Cycle  in  the  Mosquito. — As  already  explained,  this 
starts  from  the  gametocytes.  After  the  blood  is  shed,  or  after  it 
is  swallowed  by  the  mosquito,  two  important  phenomena  occur, 
namely,  (a)  the  full  development  of  the  sexual  cells  or  gameto- 
cytes, and  (b)  the  impregnation  of  the  female  (Plate  V.,  Fig. 
21  tn-y).  If  the  blood  from  a  case  of  malignant  infection  be 
examined  in  a  moist  chamber,  preferably  on  a  warm  stage,  under 
the  microscope,  both  male  and  female  gametocytes  may  be  seen 
to  become  oval  and  afterwards  rounded  in  shape  (Figs.  165-167). 
Thereafter,  in  the  case  of  the  male  cell,  a  vibratile  or  dancing 
movement  of  the  pigment  granules  can  be  seen  in  the  interior,  and 
soon  several  flagella-like  structures  shoot  out  from  the  periphery 
(Fig.  168).  They  are  of  considerable  length  but  of  great  fine- 
ness, and  often  show7  a  somewhat  bulbous  extremity.  By  the 
Romanowsky  method  they  have  been  found  to  contain  a  delicate 
core  of  chromatin,  which  is  covered  by  protoplasm.  They 
represent  the  male  cells  proper,  that  is,  they  are  sperm-cells  or 
spermatozoa;  they  are  also  known  as  microgametes.  They 
become  detached  from  the  sphere  and  move  away  in  the 
surrounding  fluid.  In  the  female  cell,  which  has  also  assumed 
the  rounded  form,  maturation  takes  place  by  the  giving  off  of 
part  of  the  nuclear  chromatin,  this  process  corresponding  to 
the  formation  of  a  polar  body.  Impregnation  occurs  by  the 
entrance  of  a  microgamete,  the  chromatin  of  the  two  cells  after- 
wards becoming  fused.  Impregnation  was  first  observed  by 
McCallum  in  the  case  of  halteridium,  and,  he  found  that  the 
female  cell  afterwards  acquired  the  power  of  independent  move- 
ment or  became  a  "  travelling  vermicule."  He  also  observed 
the  impregnation  of  the  malignant  parasite.  The  fertilised 
female  cell  is  now  generally  spoken  of  as  a  zygote  or  ookinete. 

It  has  been  established  that  the  phenomena  just  described 
occur  within  the  stomach  of  the  mosquito,  and  that  the  fertilised 
cell  or  zygote  penetrates  the  stomach  wall  and  settles  between 
the  muscle  fibres ;  on  the  second  day  after  the  mosquito  has 
ingested  the  infected  blood  small  rounded  cells  about  6  to  8  //. 
in  diameter,  and  containing  clumps  of  pigment,  may  be  found  in 


VARIETIES  OF  THE  MALARIAL  PARASITE     593 

this  position.  (It  was  in  fact  the  character  of  the  pigment 
which  led  Ross  to  believe  that  he  had  before  him  a  stage  in  the 
development  of  the  malarial  parasite.)  A  distinct  membrane 
called  a  sporocyst  forms  around  the  zygote,  and  on  subsequent 
days  a  great  increase  in  size  takes  place,  the  cysts  coming  to 
project  from  the  surface  of  the  stomach  into  the  body  cavity. 
The  zygote  divides  into  a  number  of  cells  called  blastophores  or 
gporoMatts,  and  these  again  divide  and  form  a  large  number  of 
tili form  cells  which  have  a  radiate  arrangement;  these  were 
called  by  Ross  "germinal  rods,"  but  are  now  usually  known  as 
.<)><> rozoites  or  exotospores  (in  contradistinction  to  the  enhaemospores 
of  the  human  cycle).  The  full  development  (sporogony)  within 
the  sporocyst  occupies,  in  the  case  of  proteosoma,  about  seven 
days,  in  the  case  of  the  malarial  parasites  a  little  longer. 
When  fully  developed  the  cyst  measures  about  60  JJL  in  dia- 
meter, arid  appears  packed  with  sporozoites.  It  then  bursts,  and 
the  latter  are  set  free  in  the  body  cavity.  A  large  number  settle 
within  the  large  veneno-salivary  gland  of  the  insect,  and  are  thus 
in  a  position  to  be  injected  along  with  its  secretion  into  the 
human  subject.  The  sporozoites  enter  red  corpuscles  and  become 
ama-lnihe  as  above  described.  Daniels  found  that  in  the  case 
of  the  malignant  parasite  an  interval  of  twelve  days  at  least 
intervened  between  the  time  of  feeding  the  mosquito  and  the 
appearance  of  the  sporozoites  in  the  gland. 

It  will  thus  be  seen  that  in  the  human  subject  the  parasite 
passes  through  an  indefinite  number  of  regularly  recurring 
asexual  cycles,  with  the  giving  off  of  collateral  sexual  cells,  and 
that  in  the  mosquito  there  is  one  cycle  which  may  be  said  to 
start  with  the  impregnation  of  the  female  gamete. 

Varieties  of  the  Malarial  Parasite. — The  view  propounded 
by  Laveran  was  that  there  is  only  one  species  of  malarial 
parasite,  which  is  polymorphous,  and  presents  slight  differences 
in  structural  character  in  the  different  types  of  fever.  It  may, 
however,  now  be  accepted  that  there  are  at  least  three  distinct 
species  which  infect  the  human  subject.  Practically  all  are 
agreed  as  to  a  division  into  two  groups,  one  of  which  embraces 
the  parasites  of  the  milder  fevers — "  winter-spring  "  fevers  of 
Italian  writers — there  being  in  this  group  two  distinct  species, 
!'•>;•  the  quartan  and  tertian  types  respectively ;  whilst  the  other 
includes  the  parasites  of  the  severer  forms — " sestivo-autumnal " 
fevers,  malignant  or  pernicious  fevers  of  the  tropics,  or  irregu- 
larly remittent  fevers.  There  is  still  doubt  as  to  whether  there 
are  more  than  one  species  in  this  latter  group.  Formerly 
Italian  writers  distinguished  (1)  a  quotidian;  (2)  a  non-pig- 

38 


594  MALARIAL  FEVER 

mented  quotidian ;  and  (3)  a  malignant  tertian  parasite,  though 
the  morphological  differences  described  were  slight.  Further 
observations  have,  however,  thrown  doubt  on  this  distinction, 
and  the  evidence  rather  goes  to  show  that  there  is  a  single 
species.  Opinion  also  varies  as  to  the  cycle  of  this  parasite ; 
according  to  some  observers  it  is  twenty-four  hours,  according  to 
others  forty-eight  hours,  though  there  is  more  evidence  in 
support  of  the  latter  view  ;  and  the  term  "  malignant  tertian  " 
is  frequently  used.  The  fever  is  often  of  an  irregular  type  and 
multiple  infection  is  probably  common.  Although  the  question 
cannot  be  considered  as  finally  settled,  we  shall  speak  of  three 
species  of  human  parasites.  The  zoological  position  may  be 
shown  by  the  following  scheme,  generally  followed  by  English 
writers,  the  terminology  being  chiefly  that  of  Grass!  and 
Feletti  :— 

Family :  H.EMAJVKEBID^  (Wasielewski). 

Genus  I.  Hsemamceba.  The  mature  gametes  resemble  in  form  the 
schizonts  before  segmentation  has  occurred. 

Species  1.  ffcemamceba  Danileicski  or  halter idiuin. 
Parasite  of  pigeons,  crows,  etc. 

Species  2.  ffccmamceba  rclieta  or  proteosoma. 
Parasite  of  sparrows,  larks,  etc. 

Species  3.  Hccmamccba  malaria;. 

Parasite  of  quartan  fever  of  man. 

Species  4.  Hcemamceba  vivax. 

Parasite  of  tertian  fever  of  man. 

Genus  II.  Hsemomenas.  The  gametocytes  have  a  special  crescentic 
form. 

Species :  Hccmomenas  prcccox. 

Parasite  of  malignant  or  festive-autumnal  fever  of  man. 

In  addition  there  are  other  species  belonging  to  the  same 
family  of  blood  parasites,  which  infect  monkeys,  bats,  frogs, 
lizards,  etc.,  especially  in  malarial  regions. 

We  shall  now  give  the  chief  distinctive  characters  of  the  three 
human  parasites : — 

1.  Parasite  of  Quartan  Fever. — The  cycle  of  development  in 
man  is  seventy-two  hours,  and  produces  pyrexia  every  third  day ; 
double  or  triple  infection  may,  however,  occur.  In  fresh  speci- 
mens of  blood  the  outline  is  more  distinct  than  that  of  the 
tertian  parasite,  and  amoeboid  movement  is  less  marked.  Only 
the  smaller  forms  show  movement,  and  this  is  not  of  active 
character.  The  infected  red  corpuscles  do  not  become  altered 


THE  THREE  HUMAN  PARASITES  595 

in  si/A-  or  appearance,  and  the  pigment  within  the  parasite  is  in 
the  form  of  coarse  granules,  of  dark  brown  or  almost  black 
colour.  The  fully  developed  schizont  has  a  "  daisy-head " 
appearance,  dividing  by  regular  radial  segmentation  into  from 
>ix  to  twelve  merozoites,  which,  on  becoming  free,  are  rounded 
in  form. 

2.  The   Para*<f<    <>,'   Mild  Tertian  Fever. — The  cycle  of  de- 
velopment is  completed  in  forty-eight  hours,  though  a  quotidian 
type    of   fever   may    be   produced   by  double   infection.       The 
amcebuki'  have  a  less  refractile  margin  than  in  the  quartan  type, 
and  arc  thus  less  easily  distinguished  in  the  fresh  blood;  the 
anueboid   movements    are,  however,  much   more    active,   while 
longer  and  more  slender  processes  are  given  off.     The  infected 
corpuscles   become   swollen   and   pale,    and   may   show   deeply 
stained  points  by  the  Romano wsky  method — "  Schiiffner's  dots." 
The  pigment  within  the  parasite  is  fine  and  of  yellowish-brown 
tint.        The    mature    schizont    is    rather    larger    than    in    the 
quartan,  has  a  rosette  appearance,  and  gives  rise  to  from  fifteen 
to  twenty  merozoites,  though  sometimes  even  more  occur;  these 
have  a  somewhat  oval  shape. 

In  both  the  quartan  and  tertian  fevers  all  the  stages  of 
development  can  be  readily  observed  in  the  peripheral  blood. 
The  gametocytes  have  a  rounded  form  as  described  above.  ' 

3.  The  Parasite  of  Malignant  or  JKstivo-autumnal  Fever  or 
Ti-'ij-ii-nl    Mnlnriii. — The  cycle  in  the  human  subject  probably 
occupies  forty- eight  hours,  though  this  cannot  be  definitely  stated 
in  l>e  always  the  case  (vide  mpra).     The  amcebulae  in  the  red 
corpuscles  are  of  small  size,  and  their  amoeboid  movements  are 
very  active ;  they  often,  however,  pass  into    the    quiescent  ring 
form    (Fig.    163).     The   pigment  granules,  even   in  the  larger 
forms,    are   few   in   number   and   very  fine ;    the   infected   red 
corpuscles   have  a  tendency  to  shrivel  and  assume  a  deeper  or 
coppery  tint,  sometimes  they  are  swollen  and  decolorised.     The 
fully  <leveloj>ed  schizont  usually  occupies  less  than  half  the  red 
corpuscle,  and  gives  rise  to  from  six  to  twenty  merozoites,  some- 
what   irregularly  arranged    and    of   minute    size.       Schizogony 
takes  place  almost  exclusively  in    the   internal   organs,  spleen, 
etc.,  so  that,  as  a  rule,  no  sporocytes  can  be  found  in  the  blood 
taken  in  the  usual  way.     The  proportion  of  red  corpuscles  infected 
by  the  amcebula?  is  also  much  larger  in  the  internal  organs.     The 
gametocytes  have  the  crescentic  form,  as  already  described. 

Cases  of  infection  with  the  malignant  parasite  sometimes 
assume  a  pernicious  character,  and  then  the  number  of  organisms 
in  the  interior  of  the  body  may  be  enormous.  In  certain  fatal 


596  MALARIAL  FEVER 

cases  with  coma  the  cerebral  capillaries  appear  to  be  almost 
filled  with  them,  many  parasites  being  in  process  of  sporulation ; 
and  in  so-called  algid  cases,  characterised  by  great  collapse,  a 
similar  condition  has  been  found  in  the  capillaries  of  the 
omentum  and  intestines.  The  process  of  blood  destruction, 
present  in  all  malarial  fevers,  reaches  its  maximum  in  the 
malignant  class,  and  the  brown  or  black  pigment  elaborated  by 
the  parasites — in  part  after  being  taken  up  by  leucocytes,  chiefly 
of  the  mononuclear  class — becomes  deposited  in  various  organs, 
spleen,  liver,  brain,  etc.,  especially  in  the  endothelium  of 
vessels  and  the  perivascular  lymphatics.  In  the  severer  forms 
also  brownish  yellow  pigment  is  apparently  derived  from  liberated 
haemoglobin,  and  accumulates  in  various  parts,  especially  in  the 
liver  cells ;  most  of  this  latter  gives  the  reaction  of  haemosiderin. 
General  Considerations. — The  development  of  the  malarial 
parasites  in  the  mosquito  and  infection  of  the  human  subject 
through  the  bites  of  this  insect,  have,  by  the  work  of  Ross  and 
others,  as  detailed  above,  become  established  scientific  facts. 
These  facts,  moreover,  point  to  certain  definite  methods  of  pre- 
vention of  infection,  which  have  to  a  certain  extent  already  been 
practically  tested.  The  extensive  observations  recently  carried 
out  go  to  show  that  all  the  mosquitoes  which  act  as  hosts  of  the 
parasite  belong  to  the  genus  anopheles ;  of  these  there  are  a 
large  number  of  species,  and  in  at  least  eight  or  nine  the 
parasite  has  been  found.  Some  of  these  anopheles  occur  in 
England,  especially  in  regions  where  malaria  formerly  prevailed. 
The  opportunity  for  infection  from  cases  of  malaria  returning 
from  the  tropics  to  this  country  thus  exists,  and  such  infection 
has  occurred.  The  breeding  places  of  the  insects  are  chiefly, 
though  not  exclusively,  in  stagnant  pools  and  other  collections 
of  standing  water,  and  accordingly  the  removal,  where  practicable, 
by  drainage  of  such  collections  in  the  vicinity  of  centres  of  popu- 
lation, the  covering  in  of  wells,  etc.,  and  the  killing  of  the  larvae 
by  petroleum  sprinkled  on  the  water,  have  constituted  the  most 
important  measures  in  localised  areas.  This  procedure  has  been 
carried  out  in  various  places,  for  example,  in  Freetown  and 
Ismailia,  with  marked  success.  On  the  other  hand,  where  there 
are  large  populous  areas,  as  in  India,  it  has  been  found  almost 
impracticable  to  carry  out  these  measures  with  any  success,. 
Another  measure  is  the  protection  against  mosquito  bites  by 
netting,  it  being  fortunately  the  habit  of  the  anopheles  to  rarely 
become  active  before  sundown.  The  experiments  of  Sambon 
and  Low  in  the  Campagna  proved  that  individuals  using  these 
means  of  protection  may  live  in  a  highly  malarial  district  with- 


THE  PATHOLOGY  OF  MALARIA  597 

out  becoming  iufected.  The  administration  of  quinine  to 
persons  living  in  highly  malarial  regions,  in  order  to  prevent  as 
well  as  to  treat  infection,  has  also  been  recommended  and 
carried  out,  and  the  general  agreement  appears  to  be  that  in 
India  the  properly  controlled  administration  of  quinine  must, 
in  the  meantime  at  least,  be  the  chief  means  of  combating  the 
disease.  In  the  tropics  the  natives  in  large  proportion  suffer 
from  malarial  infection,  and  one  would  accordingly  expect  that 
infection  of  the  mosquitoes  in  the  neighbourhood  of  native 
settlements  would  be  common.  This  has  been  found  to  be 
;ictually  the  ca.se,  and  it  has  accordingly  been  suggested  that  the 
dwellings  of  whites  should  as  far  as  possible  be  at  some  distance 
from  the  native  centres  of  population. 

So  far  as  is  known,  none  of  the  lower  animals  have  been 
found  to  take  the  place  of  man  as  intermediate  host  to  the 
parasites  of  malaria,  but  the  possibility  of  such  being  the  case 
cannot  be  as  yet  definitely  excluded.  On  the  death  of  infected 
mosquitoes  the  exotospores  or  sporozoites  will  become  set  free, 
and  therefore  theoretically  there  is  a  possibility  that  they  may 
enter  the  human  subject  by  inhalation  or  by  some  other  means. 
\Ve  have  no  facts,  however,  to  show  that  this  really  occurs,  and 
the  evidence  already  obtained  establishes  the  bites  of  mosquitoes 
as  the  most  important  if  not  the  only  mode  of  infection. 

It  may  also  be  mentioned  as  a  scientific  fact  of  some  interest, 
though  not  bearing  on  the  natural  modes  of  infection,  that  the 
disease  can  also  be  communicated  from  one  person  to  another  by 
injecting  the  blood  containing  the  parasites.  Several  experi- 
ments of  this  kind  have  been  performed  (usually  about  J  to  1  c.c. 
of  blood  has  been  used),  and  the  result  is  more  certain  in 
intravenous  than  in  subcutaneous  injection.  In  such  cases  there 
is  an  incubation  period,  usually  of  from  seven  to  fourteen  days, 
after  which  the  fever  occurs;  the  same  type  of  fever  is  re- 
produced as  was  present  in  the  patient  from  whom  the  blood  \\a- 
taken. 

The  Pathology  of  Malaria. — While  much  work  has  been 
done  on  the  malarial  parasite,  relatively  less  attention  has  been 
directed  to  the  processes  by  whish  it  produces  its  pathogenic 
effects.  It  may  be  said  that  the  organisms  are  not  always 
equally  prevalent  in  the  circulating  blood,  and  probably  at 
certain  stages  tend  to  be  confined  in  the  solid  organs;  thus  they 
may  be  scanty  at  the  height  of  the  paroxysm.  Some  of  the 
pathogenic  effects  are  probably  associated  with  particular  stages 
in  the  life  cycle.  Thus  the  pyrexia  occurs  when  the  stage  of 
M  hi/ogony  is  actively  in  progress.  No  opinion  can  be  stated, 


598  MALARIAL  FEVER 

however,  as  to  the  cause  of  the  fever, — whether  it  is  due  to  a 
toxic  process  or  to  general  disturbance  of  metabolism.  We  can 
better  explain  the  anaemia  which  is  so  pronounced  in  cases  where 
the  disease  is  of  long  standing,  and  which  is  due  to  the  actual 
destruction  of  red  blood  corpuscles.  The  parasite  in  its  sojourn 
in  these  cells  absorbs  their  pigment  and  thus  destroys  their 
function ;  this  is  further  evidenced  by  the  activity  displayed  by 
the  red  marrow  in  its  attempts  to  make  good  the  loss  sustained 
by  the  blood.  One  of  the  most  interesting  events  in  malaria, 
and  one  that  links  it  with  bacterial  infections,  is  the  reaction  of 
the  colourless  cells  of  the  blood.  It  has  been  shown  that  during 
the  apyrexial  stages  the  total  number  of  leucocytes  may  be 
diminished,  but  that  there  is  always  an  increase  of  the  mono- 
nuclear  cells,  these  frequently  numbering  20  per  cent,  of  the 
whole,  and  sometimes  even  outnumbering  the  polymorphs. 
This  is  such  an  important  feature  that  in  cases  where  the 
parasites  themselves  cannot  be  demonstrated  in  the  blood,  the 
mononuclear  reaction  along  with  the  presence  of  pigment  in  the 
mononuclear  cells  (due  to  phagocytosis  of  pigmented  parasites), 
has  been  taken  as  evidence  that  the  case  is  really  one  of  malaria. 
The  mononuclear  reaction  is  specially  interesting  from  the  fact 
that  in  other  protozoal  diseases  an  activity  of  the  same  elements 
has  been  observed. 

The  question  of  the  possibility  of  immunity  to  malaria  being 
developed  naturally  arises,  and  this  is  especially  interesting  in 
the  light  of  the  leucocytic  reaction  which  we  have  seen  must  be 
looked  on  as  an  element  in  immunity  against  bacterial  infection. 
With  regard  to  Europeans  developing  immunity,  it  is  difficult  to 
speak.  In  such  a  malaria-stricken  region  as  the  West  Coast  of 
Africa,  the  death-rate  in  residents  of  more  than  four  years' 
standing  is  less  than  in  the  previous  years,  but  this  may  be  due 
to  the  survival  of  the  more  resistant  immigrants.  But  there  can 
be  little  doubt  that  malaria  in  the  negro  is  a  much  less  serious 
condition  than  in  the  European.  Koch  from  his  observations  in 
New  Guinea  attributes  this  to  the  infection  of  the  native  children 
leading  to  the  development  of  immunity  in  the  adult  community. 
He  found,  what  has  been  independently  noted  by  Stephens  and 
Christophers  in  West  Africa,  that  the  greater  number  of  the 
children  harboured  malarial  parasites  in  their  blood.  The  wide- 
spread presence  of  parasites  in  children  might  appear  to  preclude 
the  immunity  of  the  adult  being  due  to  survival  of  the  most 
resistant,  but  the  infant  mortality  in  these  regions  may  be  very 
high,  and  such  a  survival  may  be  the  real  explanation.  On  the 
other  hand,  Koch  states  that  while  an  immunity  appears  to  exist 


THE  PATHOLOGY  OF  MALARIA  599 

in  native  adults  in  malarial  districts,  this  is  only  true  of  those 
born  in  the  locality, — natives  coming  from  neighbouring  non- 
malarial  districts  into  the  malarial  region  being  liable  to  contract 
the  disease.  At  present  it  must  be  held  that  the  facts  available 
do  not  enal  tie  us  to  determine  the  relative  parts  played  by  the 
development  of  artificial  immunity  on  the  one  hand,  and  the 
existence  of  a  natural  immunity  on  the  other,  in  apparent 
insusceptibility  to  malaria. 

Our  knowledge  on  the  relationship  of  blackwater  fever  to 
malaria  is  also  in  an  unsatisfactory  condition.  Blackwater  fever 
is  a  condition  often  occurring,  especially  in  Europeans,  in  tropical 
countries.  It  is  characterised  by  pyrexia,  darkly-coloured  urine, 
— the  colour  being  due  to  altered  haemoglobin  pigment, — delirium 
and  collapse,  frequently  ending  in  coma  and  death.  By  some 
the  condition  has  been  looked  on  as  a  separate  disease,  by  others 
as  the  terminal  stage  of  a  severe  malaria.  With  regard  to  the 
former  view  no  special  parasite  has  yet  been  demonstrated. 
Stephens  sums  up  the  evidence  for  the  second  view  by  saying 
that  malaria,  apart  from  the  occurrence  of  blackwater  fever,  is  a 
relatively  non-fatal  disease;  that  in  the  great  majority  of  cases 
there  is  direct  or  indirect  evidence  of  the  subject  of  the  condition 
having  suffered  from  repeated  attacks  of  malaria ;  that  while  in 
all  cases  there  must  be  an  agent  at  work  causing  ha3molysis, 
there  is  evidence  that  in  many  cases  there  is  the  possibility  of 
that  agent  being  quinine.  In  a  recent  important  work,  Christo- 
phers and  Bentley  come  to  the  conclusion  that  the  essential 
feature  in  blackwater  fever  is  an  extra-cellular  destruction  of 
red  corpuscles  in  the  blood  plasma,  a  lysaemia  as  they  call  it, 
but  that  this  is  not  directly  due  to  parasitic,  osmotic,  or  chemical 
actions,  but  to  a  specific  haemolysin  arising  in  the  body  as  the 
result  of  the  repeated  blood  destruction.  They  have  shown,  for 
example,  that  the  addition  of  fresh  serum  (complement)  to  the 
red  corpuscles  of  blackwater  fever,  as  well  as  of  malarial,  patients 
may  produce  lysis,  this  apparently  being  due  to  a  substance 
corresponding  to  immune-body  united  to  the  corpuscles  in 
qui'stion.  The  development  of  this  haemolysin  (autolysin)  results 
from  the  extensive  and  repeated  destruction  of  red  corpuscles  by 
the  malarial  parasite.  Thus  though  the  latter  is  not  the 
immediate  cause  of  the  lysaemia,  which  is  the  essential  feature  of 
l>laekwater  fever,  it  is  the  means  of  inducing  the  development 
of  the  haemolysin.  If  this  view  of  the  process  is  found  to  be 
i  oiroct,  it  would  of  course  explain  the  relationship  of  malaria  to 
the  condition.  They  also  consider  that  in  the  conditions  men- 
tioned, i.e.  where  there  has  been  repeated  destruction  of  an 


GOO  MALARIAL  FEVER 

individual's  corpuscles  by  the  malarial  parasite,  the  occurrence  of 
lysaemia  may  be  precipitated  by  an  acute  attack  of  malaria 
especially  when  under  certain  circumstances  this  is  associated 
with  the  administration  of  quinine.  On  this  view,  however,  it 
still  remains  to  be  determined  whether  the  lysis  at  the  onset  of 
an  attack  of  blackwater  fever  is  due  to  a  sudden  liberation  of 
complement  or  to  some  other  cause. 

Methods  of  Examination. — The  parasites  may  be  studied  by 
examining  the  blood  in  the  fresh  condition,  or  by  permanent 
preparations.  In  the  former  case,  a  slide  and  cover-glass  having 
been  thoroughly  cleaned,  a  small  drop  of  blood  from  the  finger 
or  lobe  of  the  ear  is  caught  by  the  cover-glass,  and  allowed  to 
spread  out  between  it  and  the  slide.  It  ought  to  be  of  such  a 
size  that  only  a  thin  layer  is  formed.  A  ring  of  vaseline  is 
placed  round  the  edge  of  the  cover-glass  to  prevent  evaporation. 
For  satisfactory  examination  an  immersion  lens  is  to  be  preferred. 
The  amoeboid  movements  are  visible  at  the  ordinary  room 
temperature,  though  they  are  more  active  on  a  warm  stage. 
With  an  Abbe  condenser  a  small  aperture  of  the  diaphragm 
should  be  used. 

Permanent  preparations  are  best  made  by  means  of  dried 
films,  which  films  are  fixed  by  one  of  the  methods  already  given 
(p.  94),  or  by  placing  in  absolute  alcohol  for  five  minutes 
(Manson).  The  films  thus  prepared  and  fixed  may  be  stained 
for  two  or  three  minutes  in  a  saturated  watery  solution  of 
methylene  blue  or  in  carbol-thionin-blue  (p.  105) ;  the  solutions 
must  be  carefully  filtered  (especially  the  latter),  and  the  films 
must  be  washed  well  after  staining.  They  are  then  dried  and 
mounted  in  balsam.  In  the  case  of  thionin-blue,  sharper  results 
are  obtained  by  dehydrating  in  alcohol  aud  clearing  in  xylol 
before  mounting.  The  best  results  are,  however,  obtained  by 
one  of  the  Romanowsky  methods  as  described  on  p.  113. 

The  fact  that  in  many  cases  the  parasites  may  be  few  in 
number  led  Ross  to  devise  a  method  for  making  their  recognition 
more  easy  by  using  blood  films  of  unusual  thickness.  Here 
about  as  much  blood  as  is  used  in  a  haemoglobin  determination 
(20  c.mm.)  is  taken  on  a  slide,  and,  being  spread  out  only  so 
much  as  to  occupy  the  area  of  an  ordinary  cover-glass,  is  allowed 
to  dry.  There  is  then  dropped  on  it  by  means  of  a  glass  rod  a 
little  of  the  watery  eosin  used  in  making  up  the  Romanowsky 
dye  (vide  p.  113).  This  is  allowed  to  act  for  about  a  quarter 
of  an  hour,  and  then  very  gently  washed  off  with  distilled  water, 
The  Romanowsky  methylene-blue  solution  is  then  applied  for  a 
few  seconds  and  also  carefully  washed  off,  and  the  preparation 


METHODS  OF  EXAMINATION  601 

dried  and  mounted.  The  hemoglobin  of  the  red  corpuscles  is 
washed  out  by  the  eosin  solution,  and  the  smaller  forms  of  the 
malarial  parasite  stand  out  as  round  circles  containing  the  char- 
acteristic chromatin  dots ;  and  in  consequence  of  the  greater 
number  present  in  a  given  area  as  compared  with  an  ordinary 
preparation  their  recognition  is  very  easy.  For  the  large  forms 
of  the  parasite  Ross  has  found  it  useful  to  make  such  a  film 
and,  hemolysing  the  red  cells  with  distilled  water,  to  examine 
it  unstained.  The  presence  of  pigment  in  the  parasites  enables 
them  to  be  readily  seen. 


APPENDIX   D. 

AMCEBIC  DYSENTERY. 

IN  a  previous  chapter  it  has  been  pointed  out  that  the  term 
"  dysentery "  has  been  applied  to  a  number  of  conditions  of 
different  etiology,  and  the  relations  of  bacteria  as  causal  agents 
have  been  there  discussed  (vide  p.  384).  We  shall  here  consider 
that  variety  of  tropical  dysentery  which  is  believed  to  be  due  to 
an  amceba,  and  hence  often  known  as  amoebic  dysentery. 

Amongst  the  early  researches  on  the  relation  of  organisms  to 
dysentery  probably  the  most  important  are  those  of  Losch,  who 
noted  the  presence  and  described  the  characters  of  amoeba  in 
the  stools  of  a  person  suffering  from  the  disease,  and  considered 
that  they  were  probably  the  causal  agents.  Further  observations 
on  a  more  extended  scale  were  made  by  Kartulis  with  con- 
firmatory results,  this  observer  finding  the  same  organisms  also 
in  liver  abscesses  associated  with  dysentery.  Councilman  and 
Lafleur,  working  in  Baltimore,  showed  that  this  variety  of 
dysentery  can  be  distinguished  from  other  forms,  not  only  by 
the  presence  of  amoebae,  but  also  by  its  pathological  anatomy. 
The  intestinal  lesions,  to  which  reference  is  made  below,  are 
of  a  grave  character,  mortality  is  relatively  high,  and  recovery, 
when  it  occurs,  is  protracted  on  account  of  the  extensive  tissue 
changes.  The  subject  was,  however,  complicated  by  the  fact 
that  a  similar  organism — the  amoeba  coli — had  been  previously 
found  in  the  intestine  in  normal  conditions  and  in  other  diseases 
than  dysentery  (by  Cunningham  and  Lewis  and  others),  and 
additional  research  confirmed  these  results.  The  matter  is  still 
far  from  being  satisfactorily  cleared  up.  While  we  may  say 
that  the  pathogenic  role  of  amoebae  has  been  established,  much 
remains  to  be  done  in  determining  what  species  have  pathogenic 
properties  and  how  these  species  may  be  identified.  The 
characters  of  the  common  amoeba  of  the  colon  and  an  amoaba 
of  dysentery  were  carefully  worked  out  by  Schaudinn,  who 
recognised  them  to  be  quite  distinct  species,  and  gave  to  them 

602 


ENTAMCEBA  HISTOLYTICA  f>03 

tlie  names  of  entamoeba  coli  and  entanm-ba  hi&tolytica  re- 
spectively. We  shall  give  the  chief  points  in  his  description, 
but  it  must  be  kept  in  view  that  amcebie  of  dysentery  studied 
by  others  present  differences  in  character.  To  these  also  refer- 
ence will  be  made  below. 

l',i>f<iin<i-li<i  /tistolytica,  as  seen  in  dysenteric  stools,  occurs  in 
the  form  of  rounded,  oval,  or  pear-shaped  cells,  measuring 
12  to  r>0  //.  in  diameter  (Fig.  169,  and  Plate  VL,  Fig.  23). 
Considerable  variations  in  size  are  met  with  in  different  cases 
of  dysentery;  in  some  acute  cases  few  am<eb;e  may  exceed 
•_M)  fi  in  diameter.  When  at  rest,  a  somewhat  clear,  highly 


/  <v, 


a  b 

Fir;.  169.— Amcebse  of  dysentery. 

<i  and  f>,  amoebae  as  seen  in  the  fresh  stools,  showing  blunt  amoeboid 
processes  of  ectoplasm.  The  endoplasm  of  a  shows  a  nucleus,  three 
red  corpuscles,  and  numerous  vacuoles  ;  that  of  1>,  numerous  red 
corpuscles  and  a  few  vacuoles. 

r,  an  amoeba  as  seen  in  a  fixed  film  preparation,  showing  a  small 
rounded  nucleus  (Kmsc  and  1'asquale).  x600. 

refract! le  ectoplasm  and  a  granular  endoplasm  can  be  dis- 
tinguished, a  feature  which  differentiates  the  organism  from 
the  entamoeba  coli.  The  nucleus  is  rounded  or  oval,  and 
is  seen  with  difficulty;  its  position  is  usually  excentric,  and  is 
sometimes  quite  at  the  margin  of  the  ectoplasm.  In  the  fresh 
condition,  and  especially  when  examined  on  a  warm  stage,  the 
organism  shows  very  active  amoeboid  movements.  The  pseudo- 
podia,  which  are  quickly  protruded  and  retracted,  are  blunt 
and  apiiear  to  be  of  tough  consistence,  a  property  which 
Sehaudinn  considers  of  importance,  as  enabling  the  organism 
to  penetrate  the  mucous  membrane,  etc.  The  amoebic  move- 
ments are  often  of  an  active  kind,  and  locomotion  may  be 


604  AMCEBIC  DYSENTERY 

fairly  rapid;  and  red  corpuscles,  bacteria,  cells,  etc.  may 
often  be  seen  in  the  interior,  though  the  ingestion  of  red 
corpuscles  is  by  no  means  a  constant  feature.  The  organism 
usually  dies  and  undergoes  disintegration  in  a  comparatively 
short  time  after  being  removed  from  the  body;  the  stools 
ought  therefore  to  be  examined  in  as  fresh  a  state  as  possible. 
Multiplication  takes  place  by  simple  amitotic  division  and  also 
by  budding.  The  entamceba  coli  is  an  organism  of  about  the 
same  size.  When  at  rest  it  shows  no  differentiation  into  ecto- 
plasm and  endoplasm,  and  the  nucleus,  usually  situated  in  the 
centre,  shows  a  highly  refractile  membrane  with  chromatin 
masses  scattered  in  the  interior.  During  amoeboid  movement 
some  delicate  processes  of  ectoplasm  come  into  view. 

Both  organisms  have  now  been  shown  to  pass  into  a  resting 
stage  with  formation  of  cysts,  the  character  and  mode  of  forma- 
tion of  which  are  markedly  different  in  the  two  cases.  The  cyst 
formation  of  the  entamceba  histolytica,  as  described  by 
Schaudinn,  is  specially  seen  when  the  disease  is  in  process  of 
cure  and  the  stools  are  beginning  to  have  a  less  fluid  character. 
In  the  earliest  stage  of  the  change  the  nuclear  membrane 
becomes  broader  and  fades  into  the  protoplasm,  whilst  the 
chromatin  becomes  dispersed  through  the  endoplasm  in  the 
form  of  small  chromidia.  Buds  then  form  on  the  surface, 
and  into  these  some  of  the  chromatin  passes.  Around  these 
buds  concentric  striation  can  be  seen,  and  then  a  hyaline 
cyst  wall  is  formed,  which  is  highly  refractile  in  character. 
The  cyst  then  becomes  separated  from  the  rest  of  the  cell. 
Several  cysts  which  measure  2  to  7  ^  in  diameter  may  be  formed 
from  the  same  amoeba,  and  the  remnant  of  the  cell  undergoes 
disintegration.  These  cysts,  as  will  be  shown  below,  repre- 
sent a  resting-stage  with  high  powers  of  resistance  to  external 
agencies,  and  are  concerned  in  producing  infection  of  another 
subject.  The  cellular  changes  in  the  encysting  of  the  entamoeba 
coli  have  also  been  worked  out  by  Schaudinn.  They  are  of 
a  somewhat  complicated  character,  involving  the  formation 
of  reduction  bodies  and  copulation  of  nuclei,  but  the  ultimate 
result  is  the  formation  of  a  fairly  large  cyst,  which  contains 
eight  small  cells.  The  process  of  cyst  formation  accordingly  in 
the  two  organisms  is  of  a  widely  different  character. 

The  description  of  the  encystment  of  amoebae  from  cases  of 
dysentery  as  given  by  some  other  observers  differs  considerably 
from  that  of  Schaudinn.  In  fact,  in  the  majority  of  the  in- 
vestigations published  no  process  of  encystment  of  buds  on  the 
surface  of  the  amoeba  has  been  observed ;  on  the  contrary,  the 


CULTIVATION  605 

whole  cell  becomes  enclosed  in  a  cyst,  which  is  of  considerable 
size.  The  facts  already  ascertained  point  strongly  to  there  being 
more  than  one  pathogenic  species  which  have  not  yet  been 
satisfactorily  distinguished. 

The  whole  subject  of  the  classification  and  means  of  distinguishing  the 
species  of  pathogenic  and  non-pathogenic  amoebae  is  still  in  a  very  un- 
satisfactory state,  and  much  further  work  is  necessary.  We  may,  however, 
refer  to  some  of  the  facts  recorded.  Musgrave  and  Clegg,  working  in 

(Manila,  cultivated  amoebae  from  drinking  water  and  from  various  other 
external  sources  as  well  as  from  cases  of  dysentery,  and  found  that  they 
possessed  similar  characters.  The  cysts  as  shown  in  their  photographs 
are  of  fairly  large  size,  and  do  not  correspond  to  Schaudinn's  description. 
By  means  of  amoebse,  cultivated  from  sources  apart  from  dysentery,  they 
were  able  to  produce  dysenteric  symptoms  and  lesions  in  monkeys, 
Lesage  cultivated  amoebic  from  cases  of  dysentery  in  Saigon  and  Toulon, 
and  found  that  the  process  of  encystment  as  studied  in  agar  plates  agreed 
with  the  account  given  by  Schaudinn.  Craig,  as  the  result  of  studies  on 
amoebae  in  San  Francisco,  confirms  the  work  of  Schaudinn  with  regard  to 
E.  coli  and  E.  histolytica.  Viereck  found  an  amoeba  in  two  cases  in 
Hamburg  which  resembled  E.  coli,  from  which,  however,  it  differed  in 
its  cysts  containing  only  four  cells.  He  gave  to  it  the  name  E. 
tetragena.  Hartmann  found  the  same  organism  in  African  dysentery,  and 
was  able  by  means  of  it  to  produce  dysentery  in  cats,  though  the  disease 
was  milder  than  with  E.  histolytica.  !Noc,  working  in  Cochin-China, 
cultivated  nim.-l»:<  from  the  intestines  in  dysentery,  from  liver  abscesses 
and  from  drinking  water,  and  found  that  they  all  had  the  same 
characters.  The  process  of  encystment  was  different  from  that  described 
by  Schaudinn,  the  whole  cell  becoming  enclosed  by  the  cyst.  In 
addition  to  the  ordinary  method  of  fission,  it  formed,  numerous  small 
cells  or  merozoites  by  a  process  of  budding  within  the  protoplasm  ;  these 
afterwards  becoming  free. 

Cultivation. — Various  attempts  have  been  made  to  cultivate 
the  amoeba  of  dysentery,  and  Kartulis  considered  that  he  obtained 
growth  in  straw  infusions.  Within  recent  years  cultures  of 
amoebae  in  association  with  various  bacteria  have  been  obtained 
on  agar  media  by  various  workers,  e.g.  Lesage,  Musgrave  and 
<']'"_rLr.  X.H-,  ami  "tlirr-.  |-'.,r  this  |»iir]m-.i-  a  plain  a^ar  without 
I>eptone  is  used,  and  its  reaction  is  made  distinctly  alkaline  to 
phenolphthaleine.  The  presence  of  .bacteria  seems  to  be 
essential  for  the  growth  of  the  amoeba?,  and  it  is  found  that  some 
species  favour  growth  whilst  others  act  prejudiciously ;  amongst 
the  former  may  be  mentioned  the  sp.  choleras,  b.  subtilis,  and 
various  HUM  nters  of  the  coli  group,  though  organisms  from  a 
great  variety  of  sources  have  been  found  to  be  equally  efficient. 

In  such  cultures,  which  are  most  conveniently  made  in  Petri 
dishes,  the  stages  of  growth  and  encystment  of  the  amoebic  can 
be  readily  studied;  the  organisms  seem  to  flourish  best  at  a 


606  AMCEBIC  DYSENTERY 

temperature  of  about  25°  C.  Although  cultures  without  bacterial 
growth  have  not  been  obtained,  means  have  been  devised  to 
ensure  that  only  one  species  of  amoeba  is  present.  For  this 
purpose  Musgrave  and  Clegg  select,  by  means  of  a  low-power 
objective,  an  amoeba  well  separated  on  the  agar  plate,  place  it  in 
the  middle  of  the  field,  then  swing  into  position  a  high-power 
objective,  and,  having  ascertained  by  means  of  it  that  the  amoeba 
is  still  there,  lower  the  point  of  the  lens  on  to  the  agar.  By 
this  means  the  amoeba  may  have  been  picked  up,  and  it  may 
then  be  transferred  to  a  fresh  plate.  These  observers  consider 
suitable  bacterial  symbiosis  to  be  of  great  importance  in  in- 
creasing the  virulence  of  the  amoebae,  and  probably  to  play  an 
important  part  in  the  pathology  of  the  disease. 

Distribution  of  the  Amoebae. — As  already  stated,  they  are 
usually  found  in  large  numbers  in  the  contents  of  the  large 
intestine  in  tropical  amoebic  dysentery.  They  also,  however, 
penetrate  into  the  tissues,  where  they  appear  to  exert  a  well- 
marked  action.  In  this  disease  the  lesions  are  chiefly  in  the  large 
intestine,  especially  in  the  rectum  and  at  the  flexures,  though 
they  may  also  be  present  in  the  lower  part  of  the  ileum.  At 
first  there  are  seen  local  swellings  on  the  mucous  surface,  chiefly 
due  to  a  sort  of  inflammatory  gelatinous  oedema  with  little 
leucocytic  infiltration ;  soon,  however,  the  mucous  membrane 
becomes  partially  ulcerated,  more  or  less  extensive  necrosis  of 
the  subjacent  tissues  occurs,  and  gangrenous  sloughs  result.  The 
ulcers  thus  come  to  have  irregular  and  overhanging  margins,  and 
the  excavation  below  is  often  of  wider  extent  than  the  aperture 
in  the  mucous  membrane.  The  amoebae  are  found  in  the  mucous 
membrane  when  ulcers  are  being  formed,  but  their  most 
characteristic  site  is  beyond  the  ulcerated  area,  where  they  may 
be  seen  penetrating  deeply  into  the  submucous  and  even  into 
the  muscular  coats.  In  these  positions  they  may  be  unattended 
by  any  other  organisms,  and  the  tissues  around  them  show 
cedematous  swelling  and  more  or  less  necrotic  change  without 
much  accompanying  cellular  reaction  beyond  a  certain  amount 
of  swelling  and  proliferation  of  the  connective  tissue  cells.  This 
action  of  the  amoeba  on  the  tissues  explains  the  character  of 
the  ulcers  as  just  described.  These  lesions  are  considered  to  be 
characteristic  of  amoebic  dysentery. 

As  a  complication  of  this  form  of  dysentery,  liver  abscesses 
are  of  comparatively  common  occurrence.  They  are  usually 
single  and  of  large  size ;  sometimes  there  are  more  than  one,  and 
occasionally  numerous  small  ones  may  be  present.  The  contents 
are  usually  a  thick  pinkish  fluid  of  somewhat  slimy  consistence, 


EXPERIMENTAL  INOCULATION 


607 


and  are  largely  constituted  by  necrosed  and  liquefied  tissue  with 

admixture  of  blood  in  varying  amount.     Microscopic  examination 

shows  chiefly  necrosed  and  granular  cells  and  debris  resulting 

from   their  disintegration,  whereas  ordinary  pus  corpuscles  are 

-rant y  or  may  be  practically  absent.     In  such  abscesses  associated 

with  dysentery  the  amoebae  are  usually  to  be  found,  and  not 

infrequently  are    the   only   organisms    present,   no   cultures   of 

bacteria  being  obtainable  by  the  ordinary  methods  (Fig.  170). 

They  are  most  numerous   at   the  spreading   margin,  and   this 

probably  explains   a    fact 

I  "tin  ted    out    by  Manson, 

that   examination    of    the 

contents  first  removed  may 

give    a     negative    result, 

while  they  may  be  detected 

in  the  discharge  a  day  or 

two  later.    The  action  here 

on    the    tissues    is    of    an 

analogous  nature,  namely, 

a  necrosis  with  softening 

and    partial    liquefaction, 

attended    by   little    or  no 

suppurative  change.     The 

anni'baj    have    also    been 

found  in  the  sputum  when 

a  liver  abscess  has  ruin  FIG.  170.— Section  of  wall  of  liver  absce-ss, 

showing  an  amoeba  of  spherical  form 
with  vacuolated  protoplasm.  From  a 
case  published  by  Major  D.  G.  Marshall. 
xlOOO. 


into 


thp 

not  very  infrequently 
happens.  Kartulis  records 
two  cases  of  brain  abscess 

occurring  secondarily  to  dysentery  in  which  numerous  amoeba; 
were  present. 

Experimental  Inoculation.  —  The  anatomical  changes  in 
dysentery,  as  above  described,  give  strong  presumptive  evidence 
as  to  the  causal  relationship  of  the  amoebae,  and  practically  con- 
clusive evidence  is  afforded  by  animal  exjjeriments.  Dysentery 
occurs  occasionally  in  animals,  e.g.  in  monkeys,  but  it  is.  of 
comparatively  rare  occurrence.  The  disease  sometimes  results 
in  the  dog  by  experimental  inoculation  with  dysenteric  material. 
Kartulis,  for  example,  records  two  cases,  in  one  of  which  liver 
abscess  was  present.  Cats  are,  however,  found  to  be  more 
susceptible,  especially  young  animals.  Dysenteric  changes  have 
been  produced  in  this  animal  by  Kartulis,  Kruse  and  Pasquale, 
and  others.  The  method  generally  adopted  is  the  introduction 


608  AMCEBIC  DYSENTERY 

of  a  small  quantity  of  mucus  from  a  dysenteric  case  into  the 
rectum.  The  resulting  disease  is  of  an  acute  character,  and 
sometimes  leads  to  a  fatal  result.  The  changes  in  the  large 
intestine  resemble  those  found  in  the  human  disease,  and 
microscopic  examination  shows  the  amoebae  penetrating  the 
wall  of  the  bowel  in  the  characteristic  manner.  Kruse  and 
Pasquale  obtained  corresponding  results  when  the  material  from 
a  liver  abscess,  containing  amoebae  without  any  other  organisms, 
was  injected.  Quincke  and  Roos  obtained  no  effects  when 
the  amoebae  were  administered  by  the  mouth,  but  they  ob- 
tained a  fatal  result  in  two  out  of  four  cases  when  the  cyst- 
like  forms  were  given.  They  also  found  that  the  cysts,  unlike 
the  amoebae,  were  still  present  even  after  the  material  had  been 
kept  for  two  or  three  weeks.  Extremely  important  confirmatory 
evidence  with  regard  to  infection  by  the  cysts  has  been  supplied 
by  experiments  of  Schaudinn.  Dysenteric  material  was  obtained 
from  China,  and  portions  of  it  which  were  found  to  contain 
the  cysts  were  thoroughly  dried.  Some  of  this  material  was 
given  with  food  to  cats  by  the  mouth,  and  typical  dysentery 
resulted,  the  amoebae  being  found  in  the  stools.  No  results 
follow  when  the  material  ingested  merely  contains  the  vegetative 
form  of  the  organism,  as  it  is  readily  destroyed  in  the  contents 
of  the  stomach. 

Musgrave  and  Clegg  produced  amoebic  colitis  in  monkeys 
by  means  of  cysts  from  cultures,  and  such  results  were  obtained 
whatever  was  the  source  of  the  amoebae, — that  is,  with  those 
obtained  from  water,  vegetables,  etc.,  as  well  as  with  those 
from  dysenteric  material.  They  also  produced  liver  abscess 
by  direct  injection  into  the  liver,  and  in  some  instances  only 
amoebae  were  present  in  the  abscesses. 

Investigations  with  regard  to  entamoeba,  coli  seem  to  show 
that  it  is  a  harmless  organism  and  that  it  is  frequently  present 
in  the  intestines  of  healthy  individuals.  Schaudinn  found  that 
in  East  Prussia  as  many  as  50  per  cent,  of  the  population  were 
infected  with  it.  The  administration  of  the  amoebae,  or  of  the 
cysts  by  the  methods  mentioned  above,  produced  no  result  in 
animals.  It  has,  however,  been  shown  that  when  the  eight- 
celled  cysts  are  swallowed  by  persons  who  are  free  from  the 
parasite  the  entamoeba  coli  appears  in  the  large  intestine  in 
a  comparatively  short  period  of  time.  It  accordingly  appears 
that  in  the  case  of  both  organisms  it  is  the  cysts  alone  which 
give  rise  to  infection.  Confirmatory  results  with  regard  to 
the  common  occurrence  of  E.  coli  were  obtained  by  Craig  in  San 
Francisco. 


METHODS  OF  EXAMINATION  609 

From  the  above  facts,  all  of  which  have  received  ample 
confirmation,  there  can  be  no  doubt  that  the  amoebae  described 
are  the  cause  of  the  form  of  dysentery  with  which  they  are 
associated.  As  already  stated,  much  information  is  still  required 
as  to  the  different  species  of  pathogenic  amoebae  and  as  to  the 
means  of  distinguishing  them,  if  this  is  possible,  from  harm- 
less forms.  In  this  connection  it  is  interesting  to  note  that 
Musgrave  and  Clegg  obtained  pathogenic  effects  with  amoebae 
resembling  the  E.  coli.  But  the  causal  relationship  of  amoebae 
to  dysentery  has  been  completely  established  by  the  anatomical 
and  experimental  evidence.  It  is  also  of  importance  to  note 
that  the  serum  of  patients  suffering  from  amoebic  dysentery 
gives  no  agglutinating  reaction  with  Shiga's  bacillus  of  dysentery 
(vide  p.  384). 

It  is  important  to  note  that  cases  of  amoebic  dysentery  have 
been  recorded  both  in  France  and  England  in  patients  who  have 
never  resided  outside  these  countries. 

Methods  of  Examination. — The  faeces  in  a  case  of  suspected 
dysentery  ought  to  be  examined  microscopically  as  soon  as 
possible  after  being  passed,  as  the  amoebae  disappear  rapidly, 
especially  when  the  reaction  becomes  acid.  A  drop  is  placed 
on  a  slide  without  the  addition  of  any  reagent,  a  cover-glass  is 
placed  over  it  but  not  pressed  down,  and  the  preparation  is 
examined  in  the  ordinary  way  or  on  a  hot  stage,  preferably  by 
the  latter  method,  as  the  •  movements  of  the  amoebae  become 
more  active,  and  it  is  difficult  to  recognise  them  when  they  are 
at  rest.  Hanging-drop  preparations  may  also  be  made  by  the 
methods  described.  Dried  films  are  not  suitable,  as  in  the 
preparation  of  these  the  amoebae  become  broken  down;  but 
wet  films  may  be  fixed  with  corrosive  sublimate  or  other 
fixative  (vide  p.  96).  In  sections  of  tissue  the  amoebae  may  be 
stained  by  methylene-blue,  by  safranin,  by  haematoxylin  and 
eosin,  and  iron  hamnatoxylin,  etc.  Benda's  method  of  staining 
with  safranin  and  light-green  is  also  a  very  suitable  one. 
Sections  are  stained  for  several  hours  in  a  saturated  solution 
of  safranin  in  aniline  oil  water  (p.  105),  they  are  then  washed 
in  water  and  decolorised  in  a  J  per  cent,  solution  of  light-green 
in  alcohol  till  most  of  the  safranin  is  discharged,  the  nuclei, 
however,  remaining  deeply  stained.  In  this  method  the  nuclei 
of  the  amoebae  are  coloured  red  (like  those  of  the  tissue  cells), 
the  protoplasm  being  of  a  purplish  tint. 


39 


APPENDIX   E. 

TRYPANOSOMIASIS— LEISHMANIOSIS—PIRO- 
PLASMOSIS. 

THE  PATHOGENIC  TKYPANOSOMES. 

THE  trypanosomata  are  protozoal  organisms  belonging  to  the 
sub-class  Flagellata,  and  many  members  of  the  genus  have  come 
to  be  recognised  as  living  in  the  blood  and  tissues  in  various 
animals  and  as  causing  important  disease  conditions.  As  long 
ago  as  1878  the  Trypanosoma  Lewisi  was  observed  infesting  the 
blood  of  rats,  and  it  has  been  found  to  be  sometimes  capable  of 
causing  death.  Other  diseases  in  which  similar  organisms  have 
been  found  are  Surra,  which  occurs  in  cattle,  horses,  and  camels 
in  India,  and  which  is  associated  with  the  Tr.  Evansi ;  Dourine, 
a  condition  affecting  horses  in  especially  the  Mediterranean 
littoral  (Tr.  equiperdum  or  Eougeti) ;  Mai  de  Caderas,  a  disease 
of  South  American  horses  (Tr.  equinum  or  Elmassiani) ;  Tse-tse 
Fly  Disease  or  Nagana,  affecting  horses  and  herbivora  in  South 
Africa  (Tr.  Brucei) ;  trypanosomiasis  of  African  cattle  (Tr. 
Theileri) ;  and — most  important  from  the  human  standpoint — 
the  trypanosomiasis  and  sleeping  sickness  of  West  and  Central 
Africa  associated  with  the  Tr.  gambiense  and  Tr.  ugandense, 
which  are  now  believed  to  be  the  same  organism.  These 
diseases  present  many  general  resemblances  to  one  another. 
They  tend  to  be  characterised  by  wasting,  cachexia,  anaemia, 
fever  often  of  an  intermittent  type  and  irregular  oedemas,  and 
frequently  have  a  fatal  result.  In  many  cases  the  infective 
agent  is  conveyed  from  a  diseased  to  a  healthy  animal  by  the 
agency  of  blood-sucking  insects. 

Morphology  and  Biology  of  the  Trypanosomata. — If  a  drop 
of  blood  containing  trypanosomes  be  examined,  the  organism 
will  be  seen  to  be  a  fusiform  mass  of  protoplasm  which  at  one 
end  passes  into  a  pointed  flagellum.  In  the  living  condition  the 
trypanosome  is  usually  actively  motile  by  an  undulatory  move- 

610 


THE  PATHOGENIC  TRYPANOSOMES  611 

incut  of  its  protoplasm  and  a  lashing  of  the  flagellum.  The 
size  varies,  but  those  mentioned  above  are  about  30  /x  long  and 
about  1*5  to  3  /n  broad.  Much  smaller  forms  exist,  however, 
and  one,  7V.  /////''"*,  which  is  7  to  10  /u,  broad  and  72  to  123  p. 
long,  has  been  described  by  Bruce.  From  the  fact  that  in 
progression  the  flagellum  is  in  front,  the  flagellated  end  is 
denominated  the  anterior  end  of  the  organism.  It  is  stated 
that  the  method  of  examining  the  fresh  blood  by  merely  allowing 
it  to  spread  itself  out  in  a  fairly  large  drop  beneath  a  cover-glass 
is  more  likely  to  reveal  the  presence  of  trypanosomes,  if  these  are 
present  in  small  numbers,  than  is  the  examination  of  stained 
specimens ;  but  the  minuter  structure  of  the  organisms  can 
best  be  studied  in  dried  preparations  stained  by  Romanowsky 
dyes  such  as  those  of  Leishman  or  Giemsa. 

For  staining  trypanosomata  (or  the  Leishman-Donovan  bodies)  in 
sections  so  as  to  bring  out  the  chromatin  structures,  Leishman  recom- 
mends the  following  method  : — Sections  of  5  /A  thickness  are  made  and 
carefully  fixed  on  slides.  The  paraffin  is  very  thoroughly  removed  by 
melting  it  before  applying  the  first  xylol,  and  then  washing  with  alter- 
nate baths  of  alcohol  and  xylol  three  or  four  times.  The  last  alcohol  is 
thoroughly  washed  oft"  by  distilled  water,  and  the  excess  of  water  is 
removed  with  cigarette  paper.  A  drop  of  fresh  blood  serum  is  then 
placed  on  the  preparation  and  allowed  to  soak  in  for  five  minutes.  The 
excess  is  removed  by  blotting,  and  the  remainder  is  allowed  to  dry  on 
the  section,  which  is  now  treated  with  a  mixture  of  two  parts  of  Leish- 
iii, in's  stain  and  three  of  distilled  water,  and  placed  in  a  Petri  dish  for 
1  to  1£  hours.  The  preparation  is  very  deeply  stained,  the  nuclei  being 
almost  black,  and  decolorisation  and  differentiation  are  effected  by  alter- 
nately applying  the  acetic  acid  and  caustic  soda  solutions  (commencing 
with  the  acid)  used  in  the  application  of  the  stain  to  ordinary  histological 
sections  (r.  p.  114),  the  effects  being  carefully  watched  with  a  low 
power.  The  essential  part  of  the  method  is  the  application  of  the  blood 
serum,  though  what  effect  this  has  is  not  known  ;  Leishman  suggests 
that  it  restores  the  normal  alkalinity  of  the  tissue. 

In  preparations  stained  by  the  above  methods  the  protoplasm 
of  trypanosomata  stains  blue,  and  in  some  species  some  parts 
are  more  intensely  coloured  than  others.  Sometimes  it  contains 
violet-coloured  granules  (chromatin  granules),  and  occasionally 
there  appears  in  it  slight  longitudinal  striation.  Two  bodies 
are  always  present  in  the  protoplasm.  Usually  near  the  middle 
there  is  an  oval  granular  body  staining  purple, — the  tropho- 
nucleus  or  macronucleus, — and  towards  the  posterior  end  is  a 
minute  intensely  stained  purple  granule  known  as  the  kineto- 
nucleus,  blepharoplast,  micronucleus,  or  centrosome  (that  this 
body  represents  the  centrosome  is  strongly  held  by  Laveran 
from  the  analogy  of  appearances  in  certain  spermatozoa  which 


6 1 2  TR  YP  ANOSOMI ASIS 

closely  resemble  trypanosomes  in  structure).  This  micronucleus 
is  often  surrounded  by  an  unstained  halo,  and  in  its  neighbour- 
hood, in  certain  species,  a  vacuole  has  been  described  as  exist- 
ing ;  this  has  been  considered  by  some  to  be  analogous  to  the 
contractile  vacuole  present  in  many  protozoa,  and  its  shape  and 
position  have  been  made  the  basis  of  specific  distinctions ; 
Laveran,  however,  thinks  it  is  an  artefact.  From  the  micro- 
nucleus  or  from  its  neighbourhood  there  arises  an  important 
structure  in  the  trypanosome, — the  undulatory  membrane. 
This  is  of  varying  breadth,  has  a  sharp  undulating  free  margin, 
and  surmounts  the  protoplasm  of  the  organism  like  a  cock's 
comb ;  it  narrows  towards  the  anterior  end  where  it  passes  into 
the  nagellum.  Motion  is  chiefly  effected  by  the  undulations  of 
this  membrane  and  of  the  nagellum.  The  latter  is  continuous 
with  the  protoplasm  of  the  body  of  the  organism ;  it  stains 
uniformly  like  it,  except  the  free  edge  which  has  the  reddish 
hue  of  the  chromatin.  In  different  species  of  trypanosomes 
variations  occur  in  shape,  in  length,  in  breadth,  in  the  position 
of  the  micronucleus  (and  therefore  in  the  length  of  the  undulat- 
ing membrane),  in  the  breath  of  the  membrane,  in  the  length  of  the 
free  part  of  the  nagellum,  in  the  shape  of  the  posterior  end,  which 
is  sometimes  blunt,  sometimes  sharp,  and  in  the  presence  or 
absence  of  free  chromatin  granules  in  the  protoplasm. 

Multiplication  in  the  body  fluids  ordinarily  occurs  by  longi- 
tudinal, amitotic  division  (see  Fig.  171).  First  of  all  the  micro- 
nucleus  divides,  sometimes  transversely,  sometimes  longitudin- 
ally, then  the  macronucleus  and  undulating  membrane,  and  lastly 
the  protoplasm.  In  some  species  the  root  of  the  nagellum  only 
divides,  so  that  in  the  young  trypanosomes  the  nagellum  is  short 
and  subsequently  increases  in  length  (Tr.  Lewisi) ;  usually  the 
whole  nagellum  takes  part  in  the  general  splitting  of  the 
organism. 

In  the  cases  of  several  of  the  trypanosomata  it  has  been 
found  possible  to  cultivate  them  outside  the  body,  the  first  work 
here  having  been  done  by  Novy-  and  MacNeal,  who  succeeded 
with  the  Tr.  Lewisi,  Tr.  Evansi,  and  Tr.  Brucei.  They  used  a 
special  medium  (see  p.  45),  on  which  it  was  found  that  multipli- 
cation went  on  readily,  the  organisms  dividing  longitudinally  as 
in  the  tissues.  Sometimes  very  small  forms  result,  and  often 
these  are  found  in  rosettes  which  are  formed  by  a  number  of 
individuals  arranging  themselves  in  a  circle  with  the  flagella 
directed  towards  the  centre  of  the  agglomeration.  These  results 
have  been  confirmed  by  other  observers,  and  by  repeated  sub- 
cultures several  of  the  trypanosomata  named  have  been  kept 


THE  PATHOGENIC  TRYPANOSOMES  613 

alive  for  more  than  a  year,  and  when  re-introduced  into  appro- 
priate hosts  have  been  found  not  to  have  lost  their  infective 
properties. 

The  main  fact  in  the  biology  of  those  trypanosomata  with 
which  the  pathologist  is  concerned,  is  that  in  the  higher  animals 
infection  takes  place  by  the  parasite  being  transferred  from  one 
host  to  another  by  the  agency  of  biting  or  blood-sucking  insects, 
or  by  other  similar  agencies  such  as  leeches.  It  may  be  said 
that  the  mere  mechanical  transference  of  the  parasite  by,  say, 
a  blood-flocking  insect,  while  it  may  sometimes  occur,  probably 
plays  a  subsidiary  part  in  infection.  Several  instances  will  be 
given  in  which  it  is  known  that  an  insect  does  not  become 
actively  infective  until  some  days  have  elapsed  after  it  has 
sucked  the  blood  of  an  infected  animal.  The  analogy  of  the 
malarial  organisms  suggests  the  occurrence  of  a  sexual  con- 
jugation within  the  insect,  but  definite  proof  of  this  is  still 
wanting,  and,  as  Minchin  points  out,  while  we  must  admit  the 
existence  of  a  cyclic  development,  it  by  no  means  follows  that 
this  includes  a  definitely  sexual  stage,  although  many  are  of 
opinion  that  such  a  stage  does  take  place. 

The  starting-point  of  the  sexual  theory  lies  in  the  slight  differences 
in  form  which  have  been  -observed  in  the  organisms  in  the  body  fluids 
of  the  vertebrate  hosts.  Such  differences  have  been  described  in  Tr. 
Leuisi  and  Tr.  Brucei  by  Prowazek,  and  in  Tr.  ugandense  by  Minchin, 
and  have  been  made  the  basis  of  a  classification  into  three  types,  which 
are  looked  on  as  representing  male,  female,  and  indifferent  individuals. 
The  male  type  is  rather  slender  both  in  body  and  in  nucleus,  the  free 
part  of  the  flagellum  is  longer  than  the  body,  and  the  protoplasm  is 
free  from  granules  ;  the  female  is  broader,  its  nucleus  is  larger  and 
rounder,  the  undulating  membrane  narrower,  the  free  part  of  the  flagellum 
is  shorter  than  the  body,  and  the  protoplasm  contains  many  chromatin 
granules,  which  are  looked  upon  as  reserve  food  material.  The  indifferent 
individuals  present  intermediate  characters.  All  multiply  by  fission 
as  described,  and,  according  to  the  supporters  of  the  sexual  theory,  the 
indifferent  individuals  can  on  occasion  become  differentiated  into  male 
or  female  forms.  The  females  are  the  most  hardy,  and  next  come  the 
indifferent  individuals  ;  if  all  but  the  females  die  out,  these  can  undergo 
parthenogenesis,  and  representatives  of  all  three  types  can  be  again 
reproduced.  The  sexual  cycle  is  represented  as  occurring  in  the  in- 
vertebrate host.  In  Tr.  Lewisi.  according  to  Prowazek,  this  is  found 
in  the  rat  louse,  hwmaiopinus  .yrinulosus.  "When  this  insect  sucks  the 
blood  of  an  infected  rat,  copulation  occurs  by  the  male  trypanosome 
entering  tin-  female  near  the  micronucleus  and  the  various  parts  of  the 
two  individual!  becoming  fused.  A  n  on -flagellated  obkinete  results, 
which,  passing  through  a  spindle-shaped  grcgarinc-like  stage  (crUhidium), 
can  develop  into  a  trypauosome  in  the  stomach  of  the  louse.  A  resting- 
stage  in  an  immature  trypanosotne-like  form  is  described  as  occurring 
in  or  on  the  intestinal"  epithelium,  and  the  parasite  is  supposed  to 
reach  the  body  cavity,  and  ultimately  the  pharynx  of  the  insect,  and 


6 1 4  TR  YP  ANOSOMI ASIS 

thus  to  find  the  opportunity  for  passing  into  the  body  of  a  fresh  host. 
Minchin,  however,  has  been  unable  to  find  evidence  of  a  sexual  stage  in 
this  trypanosome. 

A  still  further  development  of  the  views  held  as  to  the  life-history  of 
the  trypanosomes  is  found  in  the  work  of  Schaudinn,  who  investigated 
the  trypanosoma  noctuce  found  in  the  owl  (athene  noctua),  and  which  is 
carried  from  bird  to  bird  by  the  common  mosquito  (culex  pipiens).  In 
the  blood  of  the  owl  is  a  halteridium  hsemamceba  showing  pigmented 
male  and  female  forms,  closely  corresponding  to  those  observed  by 
Macallum  in  the  crow.  These,  according  to  Schaudinn,  on  reaching  the 
mosquito's  stomach  undergo  ordinary  changes — the  microgametocyte 
develops  microgametes,  one  of  which  fertilises  the  macrogamete.  An 
oval  motile  ookinete  results,  and  in  the  formation  of  its  nucleus  from 
the  male  and  female  elements  a  reduction  of  chromosomes  takes  place, 
while  the  superfluous  nuclear  structures  along  with  the  pigment  are  cast 
out  of  the  cell.  In  these  ookinetes  a  differentiation  into  male,  female, 
and  indifferent  forms  can  be  recognised,  but  the  important  new  departure 
is  that  each  can  go  on  to  develop  into  a  trypanosome.  In  the  indifferent 
ookinete  a  portion  of  the  new  nucleus  breaks  off  by  a  sort  of  heteropolar 
division,  and  the  broken-off  part  becomes  the  micronucleus  or  blepharo- 
plast  of  the  trypanosome  and  gives  origin  to  the  undulatory  membrane. 
Longitudinal  division  of  these  forms  in  the  mosquito's  intestine  may 
occur,  and  here  it  may  be  said  that  Schaudinn  differed  from  other  observers 
in  holding  that  on  division  the  membrane  does  not  split,  but  that  one  of 
the  individuals  gets  its  membrane  by  this  being  laid  down  along  the  root 
of  that  already  in  existence.  Further,  a  resting-stage  of  the  trypanosome 
may  occur,  in  which  it  becomes  attached  to  the  intestinal  epithelium, 
and,  losing  more  or  less  its  flagellate  form,  may  resemble  a  gregarine. 
The  female  ookinete  is  plumper  and  contains  more  chromatin  granules 
in  its  protoplasm  than  the  male  and  indifferent  forms,  and  is  in  virtue 
of  the  reserve  material  in  its  protoplasm  much  more  resistant  than  the 
male  or  indifferent  forms ;  when  these  die  out,  as  they  do  when,  for 
instance,  the  insect  is  starved,  it  reproduces  all  three  forms  by  a  process 
of  parthenogenesis.  In  the  female  ookinete  the  smaller  nucleus  which 
goes  to  form  the  blepharoplast  of  the  indifferent  trypanosome  divides 
into  eight  small  nuclei,  all  of  which  perish,  and  the  blepharoplast  and 
membrane  are  formed  by  a  fresh  division  in  the  large  nucleus  remaining. 
In  the  male  ookinete,  which  differs  from  the  female  in  the  clearness  of 
its  protoplasm,  a  similar  heteropolar  mitosis  takes  place,  and  again  eight 
small  nuclei  are  produced.  These  evidently  represent  the  essentially  male 
element,  for  they  persist,  and  each,  appropriating  to  itself  a  portion  of 
the  cellular  protoplasm,  detaches  itself  so  that  eight  small  trypanosomes 
are  budded  off  from  the  ookinete.  This  male  ookinete  Schaudinn  holds 
to  be  homologous  with  the  microgametocyte  occurring  in  the  blood  of 
the  owl,  and  the  small  trypanosomes  are  similarly  homologous  with  the 
microgametes  formed  when  the  blood  of  the  host  reaches  the  stomach 
of  the  mosquito.  These  small  trypanosomes  and  the  male  trypano- 
somes readily  die,  probably  because,  by  a  reduction  process  in  their 
genesis,  the  assimilative  powers  of  the  larger  nucleus  have  been 
diminished.  In  degenerating  they  often  are  found  in  the  intestinal 
tract  of  the  mosquito  arranged  in  rosettes,  with  sometimes  the  anterior 
ends,  sometimes  the  posterior  ends,  directed  towards  the  centre  of  the 
rosette.  The  next  stage  of  development  takes  place  when  the  parasites 
by  the  mosquito's  bite  reach  the  blood  of  the  vertebrate  host,  and  it  is 


THE  PATHOGENIC  TRYPANOSOMES  615 

in  connection  with  this  stage  that  Schaudinn's  observations  are  very 
tar- reach  ing.  The  indifferent  forms  by  this  time,  by  repeated  longi- 
tudinal division,  have  become  small.  In  the  owl  they  attach  them- 
selves to  red  blood  corpuscles,  which  they  penetrate,  and,  assuming  a 
halteridium  form,  grow  in  size  for  twenty-four  hours  ;  they  then  leave 
the  cells,  elongate,  again  assume  the  Hagellate  form,  move  freely  in  the 
blood  till  the  next  night,  when  they  again  enter  fresh  cells.  This  cycle 
is  repeated  for  six  nights,  the  organism  gaining  in  size  with  each  sojourn 
in  a  corpuscle.  When  the  full  size  is  thus  attained,  repeated  longi- 
tudinal division  occurs,  and  when  the  smallest  forms  are  again  reached 
the  intra-cellular  cycle  recommences.  The  largest  female  forms  prob- 
ably cannot  pass  through  the  mosquito's  proboscis,  but  when  small 
forms  reach  the  owl  they  enter  the  red  corpuscles.  They  assimilate 
food  material,  and  appear  not  to  migrate  so  frequently  as  the  indifferent 
forms.  They  lose  their  capacity  of  assuming  the  free  trypanosoma 
form,  and  ultimately  their  capacity  of  migration,  so  that  they  are 
found  lying  surrounded  by  the  remains  of  the  last  cell  they  entered. 
The  male  forms  when  they  reach  the  blood  of  the  host  rapidly  die,  and 
the  microgametocytes  are  always  recruited  from  the  indifferent  forms, 
or  from  parthenogenesis  of  the  female  forms. 

It  may  be  said  that,  according  to  Schaudinn,  the  trypanosomes  gain 
access  to  the  tissues  of  the  mosquito  by  perforating  the  intestinal  wall, 
and,  passing  through  the  body  till  they  reach  the  wall  of  the  pharynx, 
they  bant  through  this,  and  are  in  a  position  to  be  ejected  when  next 
the  insect  bites. 

Such  are  the  views  put  forward  by  this  observer  on  the  cycle 
of  life-history  of  the  Tr.  noctuse.  It  will  be  recognised  that 
the  essential  point  is  the  occurrence,  both  in  the  vertebrate  and 
invertebrate  host,  of  halteridium  stages  alternating  with  those 
in  which  the  trypanosome  form  is  assumed.  It  is  evident  that, 
if  this  were  substantiated,  important  effects  would  follow  in  our 
views  as  to  the  morphology  not  only  of  the  trypanosome  group, 
but  also  of  that  to  which  the  malarial  parasites  belong. 

Certain  criticisms  of  these  results  have  been  made,  especially  by 
Novy  and  McNeal,  who  sought  confirmatory  evidence  by  means  of  their 
culture  method.  These  observers  are  of  opinion  that  the  appearances 
described  by  Schaudinn  were  due  to  his  dealing  with  a  mixed  infection 
of  the  owls  by  trypanosomes  on  the  one  hand  and  hfemamcebse  of  the 
halteridium  type  on  the  other.  They  examined  a  very  large  number  of 
different  species  of  birds,  and  established  the  fact  that  infection  with 
halteridium  parasites  on  the  one  hand  and  with  trypanosomes  on  the 
other  is  extremely  common,  and  further,  that  in  the  blood  of  the  same 
lunl  both  halteridium  and  trypanosome  infection  could  be  observed. 
The  bird  trypanosomes  could  be  readily  cultivated,  and  it  was  observed 
that  no  cultures  were  obtainable  from  birds  in  which  halteridia  were 
alone  found,  and  further,  that  when  a  trypanosome  isolated  from  a  case 
where  both  forms  of  parasite  had  been  seen  was  injected  into  a  fresh 
bird,  only  trypanosome  forms  were  found  to  develop  in  its  body.  These 
results  are,  of  course,  not  quite  conclusive,  as  the  halteridium  stage 
might  only  follow  on  the  sexual  portion  of  the  cycle. 


6 1 6  TR  YPANOSOMI ASIS 

Reference  may  here  be  made  to  the  views  put  forward  by 
Schaudinn  regarding  the  relationship  of  certain  spirilla  to  the 
trypanosomes.  In  the  athene  noctua,  besides  the  Tr.  noctuse 
already  referred  to,  there  is  a  protozoal  parasite  infesting  the 
leucocytes  known  as  spirillum  Ziemanni  or  leucocytozoon 
Ziemanni,  whose  invertebrate  host  is  also  the  culex  pipiens. 
Ziemann  had  described  the  male  and  female  forms  in  the  owl, 
and  microgametes  had  been  observed  forming  from  the  micro- 
gametocytes.  Schaudinn  observed  the  formation  of  an  ookinete 
in  the  mosquito  ;  in  certain  cases  this  ookinete  elongates,  and 
the  vermicule  rolls  itself  up  into  a  ball  with  great  proliferation 
of  the  nucleus.  Each  little  nucleus  attaches  to  itself  a  portion 
of  the  protoplasm,  and,  becoming  a  miniature  trypanosome, 
swarms  off  and  becomes  free.  These  minute  trypanosomes 
elongate  and  develop  into  typical  spirilla  by  rolling  their  ribbon- 
shaped  bodies  spirally  along  their  longitudinal  axes,  the 
individuals  possessing  male,  female,  or  indifferent  characters, 
just  as  in  Tr.  noctme.  These  spirilla  multiply  by  longitudinal 
division,  and  often  after  fission  the  two  individuals  remain 
attached  to  each  other  by  their  posterior  ends,  and  in  this  way 
there  is  made  possible  what  is  often  seen  in  spirilla,  namely,  a 
capacity  to  move  in  either  direction.  The  spirilla  often  divide 
so  frequently  that  ultimately  the  individuals  become  invisible  by 
means  of  the  microscope,  and  can  only  be  seen  when  lying  in 
clumps.  In  this  stage  Schaudinn  thinks  the  organism  would  be 
able  to  pass  through  a  Chamberland  filter,  and  this  may  be  a 
very  important  observation,  as  throwing  light  on  the  etiology  of 
certain  diseases,  such  as  yellow  fever,  in  which  no  visible  parasites 
have  been  found. 

Schaudinn's  views  on  the  trypanosomal  characteristics  of  Sp.  Ziemanni 
raised  important  questions  regarding  the  morphology  of  other  similar 
forms  which  have  been  long  familar,  such  as  Sp.  Obermeieri,  and 
also  of  the  Spirochrete  pallida  which  Schaudinn  himself  discovered. 
It  is  as  yet  too  soon  to  express  any  opinion  on  the  ultimate  effect  of 
these  views.  According  to  Schaudinn,  a  trypanosomal  spirillum  consists 
of  a  central  thread  which  represents  the  posterior  nucleus  of  the 
trypanosome  ;  round  this  thread  the  undulating  membrane  is  spirally 
wound,  and  the  principal  nucleus  is  represented  by  minute  chromatin 
dots  sometimes  seen  in  the  course  of  the  spiral.  Whether  all  spirilla 
have  this  structure  must  be  left  for  future  investigation  to  determine. 
Difficulties  arise  with  regard  to  the  significance  of  an  undulatory 
membrane  as  a  spirillary  characteristic,  and  also  with  regard  to  the 
terminal  flagellum  which  Schaudinn  himself  found  in  Spiroclisete  pallida, 
and  which  he  had  previously  thought  did  not  occur  in  protozoal  spirilla. 
It  is  possible  that  two  groups  of  organisms  have  hitherto  been  classed 
together  under  the  name  spirillum,  and  that  one  of  these  may  still 
have  to  be  placed  with  the  bacteria. 


TKYI'ANOSOMA    LKWTSl  617 

We  now  pass  to  consider  in  detail  some  of  the  more  important 
trypanosomes. 

Trypanosoma  Lewis!.— In  1878  Lewis  described  in  rats  in  India  the 
occurrence  of  the  parasite  which  now  goes  by  his  name,  and  since  that  time 
this  trypanosome  has  been  found  to  be  very  common  in  the  blood  of  rats 
all  over  the  world,  though  the  percentage  of  animals  affected  varies  in 
different  localities.  Though  the  organism  has  no  importance  from  the 
standpoint  of  human  pathology,  several  significant  points  arise  in  con- 
nection with  it.  The  condition  in  the  rat  is  of  great  interest,  as,  though 
the  infection  runs  a  very  definite  course,  it  is  very  rarely  fatal  ;  in  fact, 
many  observers  have  been  unable  to  produce  death  by  infecting  even 
large  series  of  animals.  There  is,  however,  little  doubt  that  a  fatal  issue 
does  occur  sometimes  in  young  individuals,  especially  when  these  are 
infected  with  strains  of  the  organism  imported  from  other  localities. 
The  trypanosome,  which  is  actively  motile,  is  of  the  usual  length  but  is 
somewhat  narrow,  and  its  protoplasm  does  not  contain  any  granules.  It 
multiplies  by  fission,  of  which  Laveran  describes  two  varieties.  In  one, 
the  organism  splits  longitudinally  and  gives  rise  to  smaller  individuals 
than  the  parent.  In  the  other,  the  trypanosome  loses  its  ordinary  shape 
and  becomes  more  oval  :  nuclear  division,  which  is  often  multiple,  then 
takes  place,  and  on  subsequent  division  of  the  protoplasm  a  number  of 
small  flagellate  organisms  result ;  these  last  may  attain  the  full  form  and 
size  before  dividing  again,  or  they  may  divide  when  still  small.  When  a 
rat  is  infected  by  injection  into  the  peritoneum,  active  multiplication  goes 
on  in  the  cavity  for  a  few  days  and  then  comes  to  an  end.  Very  soon 
after  infection  the  organisms  begin  to  appear  in  the  blood  and  there 
rapid  multiplication  occurs,  the  extent  of  which  is  sometimes  so  great 
that  the  trypanosomes  may  seem  to  equal  the  red  blood  corpuscles  in 
number.  The  animal  usually  shows  no  symptoms  of  illness.  The 
infection  goes  on  for  about  two  months,  and  then  the  organisms  gradually 
disappear  from  the  blood.  In  the  great  majority  of  cases  the  rat  is  now 
immune  against  fresh  infection.  If  trypanosomes  be  introduced  into  its 
peritoneum  they  are,  according  to  Laveran,  taken  up  by  mononucleate 
phagocytes  and  destroyed.  The  serum  of  a  rat  which  has  been  infected 
shows  agglutinating  capacities  towards  the  trypanosomes,  causing  them  to 
agglomerate  in  rosettes  in  which  the  flagella  are  directed  outwards,  and 
tlie  serum  of  immune  rats  has  a  certain  degree  of  protective  action  if 
injected  along  with  the  organism  into  a  susceptible  animal.  As  has 
already  been  noted,  this  trypanosome  has  been  cultivated  on  artificial 
media,  on  which  it  multiplies  freely,  large  numbers  of  small  forms  being 
often  produced.  The-c  when  injected  into  rats  give  rise  to  the  usual 
infection,  but  not  so  rapidly  as  when  blood  from  an  infected  animal  is 
used.  The  organism  multiplies  at  the  body  temperature,  but  a  lower 
temperature  is  ] .referable,  and  at  20°  C.  Novy  and  McNeal  succeeded  in 
carrying  a  growth  through  many  sub-cultures.  The  trypanosome  is  very 
resistant  to  cooling,  and  has  been  exposed  for  fifteen  minutes  to  the 
temperature  of  liquid  air  (-191°C.)  without  being  killed.  With 
regard  to  this  infection  Minchin  and  Thomson  have  shown  that  the  rat 
flea,  eeratoftkyttwfaueiatut  transmits  the  parasite  by  the  cyclical  method 
(mechanical  infection  not  having  been  proved).  The  flea  becomes 
infective  about  a  week  after  biting,  and  remains  infective  for  a  long 
period,—  possibly  for  the  rest  of  its  life.  Infection  may  also  take  place 
through  another  species  of  flea  and  through  a  louse. 


6 1 8  TRYPANOSOMI ASIS 

Nagana  or  Tse-tse  Fly  Disease.  —This  is  a  disease  affecting 
under  natural  conditions  chiefly  horses,  cattle,  and  dogs;  it  is 
prevalent  especially  in  certain  regions  of  South  Africa,  though 
it  probably  may  occur  elsewhere.  In  the  horse  the  chief 
symptoms  are  the  following : — The  animal  is  observed  to  be 
out  of  condition,  its  coat  stares,  it  has  a  watery  discharge 
from  the  eyes  and  nose,  and  the  temperature  is  elevated ; 
swellings  appear  on  the  under  surface  of  the  abdomen  and  in 
the  legs;  it  gradually  becomes  extremely  emaciated  and 
anaemic,  and  dies  after  an  illness  of  from  two  or  three  weeks  to 
two  or  three  months.  In  other  animals  the  symptoms  are  of 
the  same  order,  though  the  duration  of  the  disease  varies  much ; 
thus  in  the  dog  the  illness  does  not  last  more  than  one  or  two 
weeks,  while  in  cattle  it  may  continue  for  six  months.  It  is 
doubtful  whether  a  domestic  animal  attacked  by  the  disease 
ever  recovers.  The  popular  idea  regarding  the  etiology  of  the 
disease  was  that  it  was  contracted  by  animals  passing  through 
certain  rather  restricted  and  sharply  denned  areas  or  belts 
characterised  by  heat  and  damp,  usually  lying  beside  rivers, 
and  always  infested  by  the  tse-tse  fly  (glossina  morsitans),  to  the 
bite  of  which  the  disease  was  attributed ;  in  this  connection  it 
is  important  to  note  that  though  man  is  frequently  bitten  by 
the  tse-tse  fly  he  does  not  contract  nagana.  Modern  know- 
ledge on  the  subject  dates  from  the  discovery  made  by  Bruce 
in  1894  that  the  blood  of  animals  suffering  from  nagana 
swarmed  with  a  trypanosome  now  known  as  the  Tr.  Brucei, 
and  in  1895  he  was  instructed  by  the  Governor  of  Natal  to 
undertake  the  investigation  which  led  him  to  work  out  the  true 
etiology  of  the  disease.  It  may  be  said  that  this  research 
forms  the  starting-point  of  the  important  work  done  during 
the  last  decade  with  regard  to  infections  by  trypanosomes. 
In  his  earlier  work  Bruce  found  that  the  parasite  was  present 
in  the  blood  of  every  animal  suffering  from  nagana  and  absent 
from  the  blood  of  healthy  animals  in  the  affected  districts; 
further,  that  the  fever  which  marks  the  onset  of  the  disease  was 
accompanied  by  the  appearance  of  the  trypanosome  in  the 
blood ;  and  finally,  that  the  transference  of  the  smallest 
quantity  of  blood  from  an  affected  to  a  healthy  animal  origin- 
ated the  disease.  He  then  proceeded  to  investigate  the  part 
played  by  the  tse-tse  fly  in  the  condition.  He  found  that  if 
flies  taken  from  the  fly  belt  were  transported  to  a  place  where 
nagana  did  not  occur,  kept  for  a  few  days,  and  then  allowed  to 
bite  susceptible  animals,  the  latter  did  not  contract  the  disease — 
this  result  showing  that  it  was  not,  as  had  been  supposed  by 


NAG  ANA  OR  TSE-TSE  FLY  DISEASE  619 

some,  a  poison  natural  to  the  insect  which  was  the  pathogenic 
agent.  But  if  such  a  fly  was  allowed  to  bite  a  dog  suffering 
from  the  disease  and  then  to  bite  a  healthy  dog,  the  latter 
contracted  the  malady  and  abundant  trypanosomes  were  found 
in  its  blood.  Again,  threads  dipped  in  the  blood  of  an  infected 
animal  and  allowed  to  dry  caused  the  disease  in  healthy  animals 
up  to,  but  rarely  beyond,  twenty-four  hours  after  being  dried  ;  if, 
however,  the  blood  were  kept  moist,  then  it  retained  its  infective- 
ness  up  to  between  four  and  seven  days ;  up  to  forty-six  hours 
living  trypanosomes  could  be  seen  in  the  tube  of  the  fly's  proboscis. 
This  corresponds  roughly  with  what  was  found  regarding  the 
limits  of  the  infectiveness  of  the  fly,  in  that  twenty-four  hours 
after  it  has  been  fed  on  an  infected  animal  its  bite  is  usually  in- 
nocuous.1 Further,  Bruce  showed  that  infection  did  not  occur  by 
any  food  or  water  partaken  of  by  an  animal  while  going  through  a 
fly  belt,  for  he  took  horses  through  such  a  region  without  allowing 
them  to  eat  or  drink,  and  found  that  they  still  contracted  the 
infection,  if  during  their  few  hours'  journey  through  the  belt 
they  had  been  bitten  by  the  tse-tse  fly.  Finally,  he  showed 
that  if  flies  were  taken  from  an  infected  area  to  a  healthy  one 
a  few  miles  off  and  allowed  at  once  to  bite  infected  animals,  the 
latter  contracted  nagana. 

By  those  experiments  it  was  thus  determined  that  nagana 
could  be  transmitted  by  the  blood  of  the  infected  animal,  that 
is,  without  the  agency  of  the  fly ;  that  the  latter  had  no  inherent 
power  to  produce  the  disease ;  that  it  could,  however,  by 
successively  biting  infected  and  healthy  animals  transmit  the 
disease  to  the  latter;  and  that  specimens  of  the  insect  caught  in 
infected  areas  harboured  the  parasite  and  were  thus  infective. 
The  question  remained  as  to  how  the  flies  might  become  infected 
in  nature.  It  had  been  observed  that  in  districts  where  the 
tse-tse  fly  lived  the  prevalence  of  the  disease  in  imported  animals 
was  related  to  the  presence  in  the  locality  of  wild  herbivora. 
ISniee  now  found  that,  if  considerable  amounts  of  the  blood 
of  the  latter  were  taken  to  another  locality  and  injected  into 
dogs,  these  in  a  proportion  of  cases  contracted  nagana,  and  from 
this  he  deduced  that  the  wild  animals  harboured  the  parasites 
in  small  numbers  in  their  blood  and  thus  kept  up  the 
possibility  of  infection.  A  further  fact  was  that  other  blood- 
sucking flies  besides  the  tse-tse  appeared  incapable  of  acting  as 
carriers  of  infection.  Bruce's  work  as  a  whole  pointed  to  the 

1  This  observation  probably  only  applies  to  infection  so  far  as  this  may 
l»r  merely  mechanical.  There  is  evidence  that  a  cyclic  development 
mvurs  iii  glossina.  and  that  thus  after  an  interval  its  bite  is  again  infective. 


620 


TRYPANOSOMIASIS 


trypanosome  as  the  cause  of  nagana,  and  this  has  since  been  finally 
established  by  the  origination  of  the  disease  by  artificial  cultures 
of  the  organism. 

The  Tr.  Brucei  (Fig.  171),  according  to  Laveran,  measures  in 
the  horse  from  28  to  33  //.long  and  from  1'5  to  2 '5  /x  broad; 
in  the  rat  and  dog  it  is  somewhat  shorter.  It  is  motile,  but  its 
activity  is  less  than  that  of  Tr.  Lewisi.  When  stained  it  presents 
the  usual  appearances;  its  posterior  end  is  usually  blunt,  and 


FIG.  171. — Trypansoma  Brucei  from  blood  of  infected  rat.  Note  in 
two  of  the  organisms  commencing  division  of  micromicleus  and  undu- 
lating membrane,  x  1000. 

the  body  often  contains  granules  in  the  anterior  portion  of  its 
protoplasm.  It  divides  longitudinally,  and,  accordng  to  Brad- 
ford and  Plimmer,  a  form  of  longitudinal  conjugation  occurs  in 
the  blood.  According  to  the  same  observers,  it  can  be  kept  alive 
for  five  to  six  days  in  blood  outside  the  body.  It  is  less  resistant 
to  the  action  of  cold  than  Tr.  Lewisi,  perishing  in  a  few  days 
at  5  to  7°  C.,  but,  like  the  other  organism,  it  can  withstand  short 
.exposures  to  temperatures  down  to  -191°  C. ;  it  is  quickly 


TIIYPANOSOMA  OF  SLEEPING  SICKNESS      621 

killed  at  44  to  45°  C.  Novy  and  McNeal  succeeded  in  cultivat- 
ing this  trypanosome  also,  though  here  it  was  very  difficult  to 
obtain  a  first  growth  from  the  blood  on  their  blood-agar  medium  ; 
once  >t!irtrd,  however,  it  was  kept  alive  through  many  sub- 
cultures, the  optimum  temperature  of  growth  being  25°  C.,  and 
it  was  from  these  sub-cultures  that  the  infection  was  obtained 
which  definitely  proved  the  organism  to  be  the  cause  of  the 
disease.  In  cultures,  as  with  Tr.  Lewisi,  short  forms  occur,  and 
there  is  sometimes  a  rosette  formation  with  the  flagella  directed 
outwards ;  agglutination  phenomena  are  also  observable  in 
defibrinated  blood.  Under  unfavourable  conditions  involution 
forms  occur,  the  organism  dividing  frequently  to  form  round 
flagellated  individuals. 

Nearly  all  laboratory  animals  are  susceptible  to  infection, 
and  the  duration  of  the  illness  corresponds  to  what  has  been 
observed  in  the  natural  infection  of  these  animals.  The  rat  has 
been  largely  used  for  experiment  and  usually  succumbs  in  about 
ten  days,  there  being  very  few  symptoms  up  till  a  few  hours 
before  death.  A  very  important  fact  has  been  observed  with 
regard  to  this  animal,  namely,  that  individuals  which  have  gone 
through  infection  with  Tr.  Lewisi  and  which  are  immune  are 
still  susceptible  to  the  Tr.  Brucei ;  from  this  it  has  been  deduced 
that  the  two  organisms  are  to  be  looked  on  as  distinct  species. 

Trypanosoma  of  Sleeping  Sickness. — Since  the  year  1800 
the  disease  called  sleeping  sickness,  sleeping  dropsy,  or  negro 
lethargy  has  been  recognised  as  prevailing  on  the  West  Coast  of 
Africa  from  the  Senegal  to  Lagos,  and  in  the  parts  lying  behind 
the  coast  between  these  regions.  It  has  also  been  found  to  be 
rife  from  Cameroon  to  Angola  and  in  the  Congo  valley,  and  to  a 
less  extent  up  the  Niger  and  its  tributaries.  In  1901  it  began 
to  appear  in  the  Uganda  Protectorate,  and  it  is  in  that  region 
that  the  investigations  have  been  carried  on  which  have  led  to  a 
knowledge  of  its  cause ;  here  it  has  wrought  very  serious  havoc 
amongst  the  native  population.  It  is  characterised  in  the  early 
stages  by  a  change  in  disposition  leading  to  moroseness,  apathy, 
disinclination  for  work  or  exertion,  and  slowness  of  speech  and 
gait.  There  may  be  headache,  indefinite  pains  about  the  body, 
the  evening  temperature  may  be  elevated  several  degrees,  the 
pulse  tends  to  be  soft  and  rapid,  and  in  a  very  large  number  of 
cases  the  superficial  glands  of  the  body  are  enlarged.  In  a 
rapid  case  the  lethargy  becomes  more  pronounced  ;  fine  tremors, 
especially  of  the  tongue  and  arms,  develop ;  progressive  emaci- 
ation occurs ;  blood  changes  appear,  consisting  of  a  progressive 
diminution  of  the  red  cells  and  of  the  haemoglobin,  and  of  a 


622  TRYPANOSOMIASIS 

lymphocytosis  in  which  the  percentage  of  both  the  large  and 
small  mononuclear  cells  is  increased,  so  that  the  former  may 
constitute  from  20  to  30  and  the  latter  from  30  to  40  per  cent, 
of  all  the  white  cells  present.  As  the  disease  progresses  the 
drowsiness  increases  till  it  deepens  into  a  coma  from  which  the 
individual  cannot  be  roused.  Often  during  the  disease  there 
occur  irregular  cedematous  patches  on  the  skin,  and  sometimes 
erythematous  eruptions,  and  effusions  into  the  serous  cavities. 
Not  every  case  runs  a  progressively  advancing  course.  Some- 
times along  with  enlargement  of  glands  the  chief  early  feature 
is  the  occurrence  from  time  to  time  of  attacks  of  fever  which 
may  be  mistaken  for  malaria,  and  from  these  apparently  com- 
plete recovery  may  take  place ;  recurrence,  however,  follows  as 
a  rule,  and  ultimately  the  typical  terminal  phenomena  may 
commence.  Such  cases  may  go  on  for  years,  and  it  is  probable 
that  many  patients  die  of  pneumonia  without  exhibiting  typical 
manifestations  of  the  malady  from  which  they  really  suffer. 
The  disease  is  an  extremely  fatal  condition,  and  probably  no 
case  where  the  actual  lethargy  is  developed  ever  recovers. 

On  considering  the  disease  from  the  standpoint  of  pathological 
anatomy  there  is  little  to  be  said.  As  Mott  described,  the  most 
striking  feature  is  the  presence  of  a  chronic  meningo-encephalitis 
and  meningo-myelitis.  The  pia-arachnoid  is  sometimes  opaque 
and  slightly  thickened  and  may  be  adherent  to  the  brain,  and 
its  vessels  usually  show  some  congestion.  The  sub-arachnoid 
fluid  is  sometimes  in  excess  and  occasionally  may  even  be  puru- 
lent. The  membranes  of  the  spinal  cord  show  similar  changes. 
The  chief  other  feature  is  the  presence  of  enlarged  lymphatic 
glands  in  the  body,  but  otherwise  there  is  nothing  special  to 
note.  With  regard  to  the  microscopic  changes,  the  chief  feature, 
according  to  Mott,  is  a  proliferation  and  overgrowth  of  the 
neuroglia  cells,  especially  of  those  which  are  related  to  the  sub- 
arachnoid  space  and  the  perivascular  lymph  spaces,  with 
accumulation  and  probably  proliferation  of  lymphocytes  in  the 
meshwork.  He  further  points  out  that  the  changes  in  the 
lymph  glands  are  of  similar  nature  and  resemble  the  infiltration 
of  the  perivascular  lymphatics  of  the  central  nervous  system. 
These  changes  are  specially  significant  in  view  of  the  lympho- 
cytosis present  in  the  blood,  which  has  already  been  noted,  and 
which  so  often  occurs  in  protozoal  infections.  In  the  nervous 
structures  there  is  comparatively  little  change,  there  being 
merely,  according  to  Mott,  some  atrophy  of  the  dendrons  of  the 
nerve  cells,  a  diminution  of  Nissl's  granules,  and  an  excentricity 
of  the  nucleus. 


TRYPANOSOMA  OF  SLEEPING  SICKNESS      623 

7'/-t/j>anosoma  f/ambiense. — Before  going  further  we  must  refer 
to  the  observation  of  a  trypanosome  in  the  blood  of  persons  not 
i-vidi'iitly  suffering  from  sleeping  sickness.  The  first  case  of 
this  was  recorded  by  Dutton  in  1901,  the  patient  being  a 
European  then  living  at  Bathurst  on  the  Gambia.  The  progress 
of  the  disease  was  here  very  slow,  and  was  characterised  by 
general  wasting  and  weakness,  irregular  rises  of  temperature, 
local  oedemas,  congested  areas  of  the  skin,  enlargement  of  spleen, 


> 


FIG.  172.—  Trvpanosoma  garnbiense  from  blood  of  guinea-pig,      x  1000. 
See  also  Plate  VI.,  Fig.  25. 

and  increased  frequency  of  pulse  and  respiration  ;  death  occurred 
a  year  after  the  case  came  under  observation  after  an  access  of 
fever,  and  a  striking  fact  was  the  absence  of  any  gross  causal 
K'sion.  During  the  time  the  patient  was  under  observation 
trypanosomes  were  repeatedly  demonstrated  in  the  peripheral 
blood,  and  they  also  developed  in  the  bodies  of  monkeys  and 
white  rats  inoculated  with  the  blood.  Pursuing  further  in- 
quiries, Dutton  and  Todd  demonstrated  similar  parasites]  tin 
other  Europeans  and  in  several  natives  in  the  Gambia  region, 


624  TRYPANOSOMIASIS 

whilst  about  .the  same  time  Manson  reported  a  case  of  the  same 
kind  in  the  wife  of  a  missionary  on  the  Congo.  It  thus  came 
to  be  recognised  that  in  man  there  occurred  a  disease  having 
characters  somewhat  resembling  nagana  and  in  which  trypano- 
somes  could  be  demonstrated  in  the  blood,  and  this  was  usually 
referred  to  as  human  trypanosomiasis,  or  trypanosoma  fever, — 
the  trypanosome  being  named  the  Tr.  gambiense. 

Relation  of  Trypanosomes  to  Sleeping  Sickness. — Several 
views  as  to  the  etiology  of  this  disease  had  been  advanced.  A 
Portuguese  Commission  in  1902  described  a  diplococcus,  tending 
to  grow  in  chains,  which  they  isolated  from  the  cerebro-spinal 
fluid  taken  from  cases  during  life,  and  to  which  they  were 
inclined  from  the  constancy  of  its  occurrence  to  attribute  a 
causal  role.  The  seriousness  of  the  epidemic  in  Uganda  had  led  the 
Royal  Society  of  London  in  1902,  at  the  instigation  of  the  Foreign 
Office,  to  despatch  a  Commission  to  investigate  the  condition  on 
the  spot.  Soon  after  commencing  work,  Dr.  Castellani  found 
in  some  cases  in  the  cerebro-spinal  fluid,  especially  when  this 
was  centrifugalised,  living  trypanosomes  resembling  the  Tr. 
gambiense ;  he  also  found  in  80  per  cent,  of  the  cases  post  mortem 
a  coccus  resembling  if  not  identical  with  that  observed  by  the 
Portuguese  Commissioners.  At  first  Castellani  was  inclined  to 
look  on  the  presence  of  the  protozoon  as  accidental,  but  Colonel 
Bruce,  on  going  out  with  Nabarro  and  Greig  in  1903  to  pursue 
the  work  of  the  Commission,  realised  the  significance  of  the 
observation,  urged  Castellani  to  further  inquiries,  which  he 
himself  continued  after  the  departure  of  the  latter,  with  the 
result  that  in  a  series  of  examinations  carried  out  in  several 
infected  localities,  the  trypanosome  was  demonstrated  in  every 
case  of  the  disease.  This  work  formed  the  starting-point  for 
inquiries,  the  results  of  which  make  it  practically  certain  that 
the  parasite  is  the  causal  agent  of  the  condition.  The  organisms 
were  not  demonstrable  in  the  cerebro-spinal  fluid  of  patients 
dying  of  other  diseases  in  the  sleeping  sickness  area.  On  the 
other  hand,  it  was  found  that  if  cerebro-spinal  fluid  withdrawn 
from  cases  of  the  disease  was  injected  into  monkeys  (especially 
macacus  rhesus),  trypanosomes  appeared  in  the  blood,  and  in 
many  cases  in  three  or  four  months  the  animals  died  of  an  ill- 
ness indistinguishable  from  sleeping  sickness,  and  with  the  para- 
sites in  the  central  nervous  system.  It  was  further  found  that  in 
the  parts  round  the  north  end  of  Victoria  Nyanza  where  sleeping 
sickness  was  rife,  the  distribution  of  the  disease  exactly  corre- 
sponded with  the  distribution  of  a  blood-sucking  insect,  the 
glossina  palpalis,  a  species  closely  allied  to  the  c/lossina  morsitans 


TKYPANOSOMA  OF  SLEEPING  SICKNESS      625 

of  nagana.  It  was  found  that,  when  one  of  these  Hies  was 
fed  on  a  sleeping  sickness  patient  and  then  allowed  to  bite  a 
monkey,  frequently  trypanosomes  appeared  in  the  animal's 
blood,  and  that  when  fresh  flies  caught  in  the  sleeping  sick- 
ness area  were  placed  on  a  monkey  a  similar  occurrence  took 
place. 

The  trypauosome  of  sleeping  sickness  is  17  to  28  //,  long  and 
1*4  to  2  JJL  broad  (Fig.  172);  when  about  to  divide  it  is  both  longer 
and  broader.  According  to  Laveran,  the  free  part  of  the  flagellum 
often  equals  a  fourth  of  the  whole  length,  but  occasionally  the 
body  protoplasm  extends  quite  to  the  end  of  the  organism. 
The  undulating  membrane  is  narrow,  and  the  posterior  end  may 
be  either  sharp  or  blunt.  The  trypanosome  contains  the  macro- 
and  micronucleus  characteristic  of  the  group,  and  the  proto- 
plasm often  shows  chromatin  granules.  Castellaui  attached  great 
importance  to  a  vacuole  often  seen  in  the  neighbourhood  of  the 
inicronucleus,  but,  as  stated  above,  Laveran  holds  this  to  be  an 
artefact.  The  organism  divides  longitudinally  in  the  usual  manner, 
HI  id  often  two  can  be  seen  to  approach  each  other  and  lie  more  or 
less  side  by  side,  but  whether  this  indicates  conjugation  or  not  is 
not  known.  The  organism  does  not  usually  long  survive  removal 
from  the  body,  but  it  has  been  found  to  be  motile  for  nineteen 
days  when  kept  on  rabbit-blood  agar  at  22°  C.  As  we  have 
said,  when  Tr.  ugandense  is  inoculated  into  monkeys  they  often 
contract  an  illness  which  ultimately  presents  the  features  of 
typical  sleeping  sickness.  Inoculation  of  other  species  of  animals 
is  not  usually  so  successful,  though  in  nearly  every  case,  e.g.  in 
the  guinea-pig,  a  proliferation  of  the  parasite,  as  indicated  by  its 
appearing  in  the  blood,  takes  place ;  but  often  either  no  disease 
occurs  or  this  runs  a  very  chronic  course.  The  relative  insuscepti- 
bility of  animals,  especially  of  the  dog,  to  the  Tr.  ugandense  is 
taken  as  evidence  that  this  organism  is  essentially  different  from 
Tr.  Brucei. 

By  means  of  microscopic  examination  the  organisms  may  be 
found  in  the  cerebro-spinal  fluid,  the  blood,  or  the  juice  of 
glands.  In  the  case  of  the  first  about  10  c.c.  of  the  fluid  is  to 
l)o  ceutrifugalised  for  fifteen  minutes  and  the  deposit  placed  under 
a  cover-glass  for  examination ;  it  is  better  to  make  a  little  cell 
on  a  slide  by  painting  a  ring  of  ordinary  embedding  paraffin, 
t'»  |ilace  the  droplet  of  fluid  in  its  centre,  and  to  support  the 
cover-glass  on  the  paraffin ;  in  this  way  injury  to  the  delicate 
structure  of  the  organism  is  avoided.  In  •  fresh  cerebro-spinal 
fluid  the  trypanosomes  can  be  seen  to  be  actively  motile ;  the 
number  in  which  they  occur  varies  very  much,  and  the  same  is 
40 


626  TRYPANOSOMIASIS 

true  to  a  greater  degree  of  the  blood,  in  which  they  are,  however, 
usually  very  scanty.  With  regard  to  the  examination  of  the 
blood,  Bruce  and  Nabarro  state  that  it  is  difficult  by  ordinary 
centrifugalisation  to  concentrate  the  organisms,  as  these  are  not 
readily  precipitated.  They  accordingly  recommend  that  the 
blood  be  mixed  with  citrate  of  sodium  solution  (equal  parts  of 
blood  and  of  a  one  per  cent,  citrate  solution)  and  centrifugalised 
for  ten  minutes,  that  the  plasma  be  removed  and  centrifugalised 
afresh  for  the  same  time,  and  that  this  be  repeated  three  times, 
the  deposit  from  each  centrifugalisation  after  the  first  being 
carefully  examined.  Greig  and  Gray  have  insisted  that  the 
examination  of  the  glands  in  a  suspected  case  forms  the  most 
ready  means  of  arriving  at  a  diagnosis,  and  this  opinion  has 
found  strong  support  from  the  work  of  Button  and  Todd.  The 
method  is  to  push  a  hypodermic  needle  into  the  gland,  suck  up 
a  little  of  the  juice,  and  blow  it  out  on  to  a  slide.  In  all  cases 
where  films  of  any  kind  are  to  be  prepared  the  staining  methods 
of  Leishman  or  Giemsa  are  to  be  recommended.  Often  in 
cerebro- spinal  fluid  and  gland  juice  the  staining  of  the  chroma- 
tin  is  difficult,  but  good  preparations  are  obtained  by  the  pro- 
cedure recommended  by  Leishman  for  studying  the  parasite  in 
sections  (p.  114). 

Greig  and  Gray  have  studied  the  trypanosome  in  the  body  of 
the  glossina.  They  found  evidence  of  its  multiplying  in  the 
stomach  of  the  insect,  and  it  also  was  seen  to  undergo  changes 
not  elsewhere  observed.  These  consisted  in  alterations  in  the 
position  of  the  micronucleus,  which  often  became  anterior  to  the 
macronucleus ;  there  also  occurred  rosettes,  consisting  of  from 
four  to  twenty  individuals  attached  by  their  posterior  extremities. 
Oval  forms  were  also  observed.  It  was  at  first  supposed  that 
monkeys  could  not  be  inoculated  with  the  trypanosomes  from 
the  stomach  of  the  fly,  but  recently  Bruce  has  succeeded  in 
originating  an  infection  in  this  wray;  probably,  however,  the 
organism  remains  alive  in  only  a  small  proportion  of  flies  biting 
an  infective  case.  Minchin  in  this  connection  has  described  in  the 
gut  of  the  fly  different  types  of  the  parasite  corresponding  with  the 
male,  female,  and  indifferent  forms  found  in  other  trypanosomes. 
This  was  confirmed  by  Koch  and  by  Klein,  who  also  found  in 
the  intestine  agglomerations  of  immature  forms  which  they 
ascribed  to  the  results  of  sexual  conjugation.  The  most  im- 
portant fact  established  by  the  last  observer  was,  however,  that 
when  Gl.  palpalis  was  allowed  to  bite  an  animal  suffering  from 
nagana  it  did  not  become  infective  for  some  days.  This  has 
been  confirmed  for  Gl.  palpalis  in  the  case  of  monkeys  suffering 


TRYPANOSOMA  OF  SLEEPING  SICKNESS      627 

from  Tr.  gambiense  by  Bruce  and  those  associated  with  him  in 
1908-9.  Here  it  was  found  that  infectivity  did  not  appear  till 
about  thirty-two  days  after  the  fly  had  fed,  and  continued  until 
at  least  seventy-five  days.  In  this  connection  certain  facts 
having  a  serious  bearing  on  the  continued  infectivity  of  a  local- 
ity have  emerged.  It  was  found  that  a  certain  island  on  Lake 
Victoria  Nyanza,  which  had  been  cleared  of  infective  natives  two 
years  previously,  still  harboured  infective  flies.  To  account  for 
this  it  must  be  supposed  either  that  the  glossina  has  an  extended 
duration  of  life  or  that  the  trypanosome  exists  among  the  wild 
animals.  As  it  has  been  found  that  cattle  may  be  infected  with 
the  parasite,  and  may  through  the  medium  of  the  fly  infect 
monkeys,  it  is  possible  that  wild  herbivora,  while  not  suffering 
in  any  serious  way  themselves,  are  the  means  of  maintaining 
infectivity.  There  is  no  definite  evidence  that,  as  Koch  supposed, 
the  crocodile  harbours  the  trypanosome. 

Early  in  the  Uganda  investigations  the  question  arose  as  to 
whether  the  trypanosoma  of  sleeping  sickness  was  different  from 
Tr.  gambiense.  This  was  forced  on  the  inquirers  by  the  fact 
that  a  very  large  proportion  of  the  natives  in  the  sleeping 
sickness  area  were  found  to  harbour  trypanosomes  in  their 
blood,  although  not  apparently  suffering  from  the  disease. 
Several  cases  were  carefully  examined  in  which  trypanosomes 
were  constantly  present  in  the  blood,  but  in  which  the  patients 
from  time  to  time  suffered  from  fever,  and  during  these  pyrexial 
periods  trypanosomes  were  found  in  the  cerebro-spinal  fluid.  It 
was  suggested  that  these  cases  were  on  the  way  to  develop  sleep- 
ing sickness.  A  very  important  observation  was  that  while  in 
sleeping  sickness  areas  a  large  proportion  of  the  native  popula- 
tion harboured  trypanosomes,  this  was  not  the  case  where  sleep- 
ing sickness  did  not  occur.  Further,  it  was  found  that 
trypanosomes  from  the  cerebro-spinal  fluid  of  sleeping  sickness 
cases  and  from  the  blood  of  persons  harbouring  trypanosomes, 
but  not  suffering  from  disease  symptoms,  gave  rise  in  monkeys 
to  the  same  group  of  chronic  effects  which  resembled  the  last 
.stages  of  the  disease  in  man.  These  facts  led  the  Commissioners 
to  incline  to  the  idea  that  trypanosoma  fever  and  sleeping  sick- 
ness are  due  to  the  same  cause,  and  represent  different  stages 
<»f  the  same  disease.  It  has  already  been  pointed  out  that  a 
fatal  termination  can  occur  in  trypanosoma  fever  by  an  acute 
febrile  attai-k  or  from  intercurreut  disease,  and  thus  the  terminal 
lethargic  stage  may  only  develop  in  a  certain  proportion  of  cases. 
Continued  observation  of  prolonged  cases  of  trypanosoma  fever, 
-both  in  Uganda  by  Greig  and  Gray,  and  in  this  country  by 


628  TRYPANOSOMIASIS 

Manson,  has  shown  that  sometimes  the  termination  of  a  case  is 
by  the  onset  of  typical  sleeping  sickness.  There  is  now  practi- 
cally no  doubt  that  the  two  conditions  are  etiologically  identical. 
The  best  authorities  are  agreed  that  morphologically  no  difference 
between  the  Tr.  gambiense  and  the  Tr.  ugandense  can  be 
recognised,  and  from  considerations  of  priority  the  former  term  is 
now  alone  employed. 

The  prevalence  of  trypanosomes  in  the  blood  of  apparently 
healthy  natives  has  raised  the  question  of  the  possibility  of 
tolerance  existing  and  of  immunity  being  established.  It  is 
possible  that  both  phenomena  occur,  that  not  every  infection 
results  in  multiplication  of  the  parasite  in  the  body  of  the 
victim,  and  that  in  certain  cases  where  multiplication  does  occur 
a  resistance  is  developed  which  enables  the  body  to  kill  the 
parasites.  The  occurrence  of  the  mononuclear  reaction  is  here 
significant ;  it  has  been  suggested  that,  when  this  resistance  is 
weak,  the  organism  gains  entrance  to  the  spinal  canal,  and  that 
then  sleeping  sickness  results. 

The  whole  of  the  recent  work  on  the  disease  is  of  the  highest 
interest  and  importance.  The  strongest  evidence  may  be  said 
to  exist  that  the  Tr.  gambiense  is  the  cause  of  sleeping 
sickness,  and  action  taken  on  this  supposition  has  had  a  very 
important  effect  in  checking  the  ravages  of  the  disease  in  Uganda, 
where  the  natives  have  been  deported  from  the  fly  areas,  and  the 
brushwood  in  which  the  insects  lodge  has  been  cut  down  in  the 
neighbourhood  of  ferries. 

Not  much  success  has  attended  remedial  efforts  in  those  suffer- 
ing from  infection.  Here  attention  has  been  chiefly  concentrated 
on  the  action  of  organic  arsenical  compounds,  the  application  of 
which  in  the  shape  of  atoxyl  was  first  recommended  by  Thomas. 
A  great  range  of  such  substances  and  also  of  aniline  derivatives 
has  been  investigated  by  Ehrlich  and  his  co-workers,  and  under 
certain  conditions  of  artificial  infection  in  animals  a  complete  or 
partial  destruction  of  the  parasites  has  followed  administration 
of  these  bodies,  but  their  application  to  natural  infections  has 
not  as  yet  met  with  decided  success.  Sufficient,  however,  is 
known  to  justify  further  investigations  of  a  similar  kind.  It 
has  been  observed  that  a  tolerance  of  such  reagents  can  be 
developed  by  the  trypanosomes,  and  this  fact  may  complicate  the 
problem  at  issue. 

Other  Pathogenic  Trypanosomes. — Apart  from  sleeping  sick- 
ness no  other  important  disease  of  man  has  been  found  to  be 
associated  with  trypanosomal  infection,  but  some  observations 
on  a  condition  observed  in  Brazil  may  be  alluded  to. 


TRYPANOSOMA  CRUZI  629 

Trypanosoma  Cruzi. — Chagas,  working  in  Brazil,  observed  this 
trypanosonir  in  a  monkey,  the  intermediate  host  being  a  hemipterous 
insect  of  tin-  genus  £'"//'//•/< //»'*.  As  this  insect  also  feeds  on  man,  the 
|ius>il.le  relationship  of  the  trypanosome  to  a  human  disease  occurring 
in  that  region  was  considered.  This  disease  affects  children,  and  is 
characterised,  by  pronounced  anaemia,  the  occurrence  of  oedema,  and 
enlargement  of  lymphatic  glands,  the  spleen  and  liver  ;  it  is  associated 
with  a  mental  condition  of  infantilism,  and  ends  in  death  with  convulsions. 
The  parasite  was  only  in  one  case  found  in  the  blood  of  infected  individuals, 
but  when  the  blood  was  injected  into  guinea-pigs,  or  into  callithrix 
monkeys,  a  definite  disease  occurred,  leading  to  death.  In  the  peripheral 
blood  in  such  animals,  besides  free  forms,  an  infection  of  the  red  blood 
corpuscles  witli  a  body  resembling  a  malarial  merozoite  was  seen,  this 
body  apparently  developing  into  a  trypanosoma-like  organism.  A  special 
development  of  the  parasite  seemed  to  occur  in  the  lungs,  the  result  of 
which  was  the  formation  of  cells  containing  eight  bodies  resembling  the 
merozoite  forms  seen  in  the  circulation,  and  analogous  to  what  Schaudinn 
described  in  the  mosquito's  stomach  in  connection  with  trypanosoma 
noctua?.  A  cycle  of  development  was  also  observed  in  the  intestinal  tube 
of  the  conorhinns,  and  cultures  were  obtained  on  Novy  and  MacNeal's 
medium. 

1 t  is  beyond  the  scope  of  this  work  to  deal  at  length  with  the 
other  diseases  of  animals  caused  by  trypanosomes.  The  chief  of 
these  have  been  mentioned  in  the  opening  paragraph,  but  it  may 
be  said  that  many  others  have  been  described  in  various  species 
of  mammals,  bin  Is,  and  fishes,  and  that  these  are  spread  either 
by  flies  or  by  leeches.  The  most  interesting  of  those  mentioned 
is  Dourine,  a  condition  resembling  in  many  ways  nagana.  It, 
ho\\rver,  presents  this  peculiarity,  that  infection  does  not  take 
place  by  an  intermediate  host,  but  apparently  directly  through 
coitus,  as  it  occurs  only  in  stallions  and  in  mares  covered  by 
theea 

In  several  of  the  trypauosomal  infections  of  animals  it  appears 
as  if,  as  in  the  case  of  Tr.  Lewisi,  the  animal  suffers  little 
inconvenience  from  the  presence  of  the  parasite  in  its  blood, 
and  the  view  has  even  been  put  forward  that  with  all  pathogenic 
tr\  -pa  i  incomes  there  exists  a  host  which  acts  as  a  "reservoir"  and 
carries  the  organism  without  being  affected  by  its  presence  more, 
for  example,  than  is  the  rat  by  Tr.  Lewisi.  Though  no  opinion 
can  be  expressed  on  this  j>oint,  it  is  necessary  to  bear  the  fact 
in  mind  that  either  natural  or  acquired  immunity  can  exist 
against  such  protozoa.  Not  only  is  this  important  from  the 
point  of  view  of  the  investigation  of  the  conditions  under  which 
sueh  tolerance  fttiaea,  but  also  from  the  bearing  which  the 
existence  of  this  tolerance  may  have  on  the  spread  in  nature  of 
the  parasites  to  a  susrrptibli-  -|  eCMfl  from  immune  animals  which 
still  liarbiuir  trypanost.mes  in  their  blood.  \Ve  are,  however, 


630  LEISHMANIOSIS 

as  yet  quite  ignorant  of  many  of  the  processes  at  work  in  the 
body  during  a  trypanosomal  infection,  and  of  the  causes  of  the 
symptoms  and  other  morbid  effects. 

LEISHMANIOSIS. 

Under  this  term  there  are  grouped  three  human  diseases,  but 
the  exact  zoological  place  of  the  parasites  among  the  protozoa 
cannot  be  said  to  be  at  present  definitely  settled.  These 
organisms  are  the  Leishmania  donovani,  associated  with  the 
human  disease,  kala-azar ;  Leishmania  infantum,  derived  from  a 
similar  disease  occurring  in  children;  and  Leishmania  tropica, 
which  has  been  found  in  a  skin  ulceration  of  widespread 
geographical  distribution.  Microscopically  the  organisms  are 
practically  identical,  but  at  present  it  is  convenient  to  look  upon 
the  three  species  as  being  distinct. 

Leishmania  Donovani. — Leishman  noticed  in  several  soldiers 
invalided  from  India  for  remittent  fever  and  cachexia  that  the 
most  careful  examination  of  the  blood  failed  to  reveal  the 
presence  of  the  malarial  parasite.  From  the  fact  that  such 
patients  had  almost  invariably  been  quartered  during  their 
service  at  Dum-Dum,  an  unhealthy  cantonment  near  Calcutta, 
he  suspected  he  had  to  deal  with  an  undescribed  disease.  In 
1900  he  noticed  in  the  spleen  of  such  a  case  peculiar  bodies 
which,  from  comparison  with  certain  appearances  found  in 
degenerating  forms  of  Tr.  Brucei,  he  suggested  might  be 
trypanosomes,  and  on  publishing  his  observations  in  1903  he 
put  forward  the  view  that  trypanosomiasis  might  prevail  in 
India  and  account  for  the  aberrant  cases  of  cachexial  fever  met 
with  there.  Soon  after  Leishman's  paper  appeared,  his  observa- 
tions were  confirmed  in  India  by  Donovan,  and  the  bodies 
associated  with  the  disease  are  usually  called  the  "  Leishman  "  or 
the  "  Leishman-Donovan  "  bodies.  They  were  found  by  Bentley, 
and  later  by  Rogers,  in  the  disease  known  in  Assam  as  kala-azar, 
the  pathology  of  which  had  long  puzzled  those  who  had  worked 
at  it,  from  the  fact  that,  while  it  resembled  malaria  in  many 
ways,  no  parasite  could  be  demonstrated  to  occur  in  those 
suffering  from  it.  This  disease  has  gone  under  various  synonyms, 
e.g.  cachetic  fever,  Dum-Dum  fever,  non-malarial  remittent 
fever,  but  is  now  recognised  as  a  single  entity. 

Kala-azar  (or  "black  disease," — so  called  from  the  hue 
assumed  by  chocolate-coloured  patients  suffering  from  it)  has 
been  known  since  1869  as  a  serious  epidemic  disease  in  Assam, 
where  it  has  spread  from  village  to  village  up  the  Brahmaputra 


LEISHMANIA  DONOVANI  631 

valley.  The  disease  is  now  known  to  occur  in  various  sub- 
tropical centres  between  the  forty-ninth  parallels — cases  where 
the  Leishman  bodies  have  been  found  having  been  met  with 
in  many  parts  of  India,  China,  the  Malay  Archipelago,  North 
Africa,  the  Soudan,  and  Arabia.  The  disease  is  characterised 
by  fever  of  a  very  irregular  type,  by  progressive  cachexia,  and  by 
anaemia  associated  with  enlargement  of  the  spleen  and  liver, 
and  often  with  ulcers  of  the  skin  and  dropsical  swellings. 
Rogers  has  pointed  out  that  there  occurs  a  leucopenia  which 
differs  from  that  of  malaria  in  that  it  is  almost  always  more 
marked, — the  leucocytes  usually  numbering  less  than  2000, — and 
further,  in  that  the  white  cells  are  always  reduced  in  greater  ratio 
than  the  red  corpuscles,  which  condition,  again,  does  not  occur  in 
malaria.  The  disease  is  chronic,  often  going  on  for  several  years, 
and,  at  any  rate  in  the  great  majority  of  cases,  has  a  fatal 
issue.  Post  mortem,  there  is  little  to  note  beyond  the  enlarge- 
ment of  the  liver  and  spleen,  but  in  the  intestine,  especially 
in  the  colon,  there  are  often  large  or  small  ulcers,  and  there  is 
evidence  of  proliferation  in  the  bone  marrow,  the  red  marrow 
encroaching  on  the  yellow. 

In  a  film  made  from  the  spleen  and  stained  by  Irishman's 
stain,  the  characteristic  bodies  can  be  readily  demonstrated 
(Fig.  173).  They  are  round,  oval,  or,  as  Christophers  has 
pointed  out,  cockle-shell  shaped,  and  usually  2*5  to  3*5  /x  in 
diameter,  though  smaller  forms  occur.  The  protoplasm  stains 
pink,  or  sometimes  slightly  bluish,  and  contains  two  bodies 
taking  on  the  bright  red  colour  of  nuclear  matter  when  stained 
by  the  Romanowsky  combination.  The  larger  stains  less 
intensely  than  the  smaller,  is  round,  oval,  heart-shaped,  or 
bilobed,  and  lies  rather  towards  the  periphery  of  the  body — in 
the  region  of  the  "  hinge "  in  the  cockle-shaped  individuals. 
The  other  chromatin  body  is  usually  rod-shaped,  and  is  set 
perpendicularly  or  at  a  tangent  to  the  larger  mass,  with  which 
only  exceptionally  it  appears  to  be  connected.  Usually  the 
protoplasm  contains  one  or  two  vacuoles.  Though  in  spleen 
smears  many  free  bodies  are  seen,  the  study  of  sections  shows 
that  ordinarily  their  position  is  intra-cellular, — the  cells  con- 
taining them  being  of  a  large  mononuclear  type  (Fig.  174).  The 
view  held  is  that  on  their  entering  the  circulation  they  are 
taken  iij>  by  the  mononuclear  leucocytes  and  by  such  cells  as 
the  endothelial  lining  of  the  splenic  sinuses  or  by  those  lining 
capillaries  or  lymphatics,  that  in  these  cells  multiplication  takes 
place — it  may  be  to  such  an  extent  as  to  rupture  the  cell, — and 
that  if  thus  the  bodies  become  free  they  are  taken  up  by  other 


632 


LEISHMANIOSIS 


cells  and  the  process  is  repeated.  The  clusters  of  bodies  some- 
times seen  in  smears  are  probably  held  together  by  the  remains 
of  ruptured  phagocytes.  In  capillaries  the  endothelial  cells  after 
phagocyting  the  bodies  probably  become  detached  from  the 
capillary  wall,  as  they  are  often  observed  free  in  the  lumen  of 
the  vessel, — this  being  well  seen  in  the  hepatic  capillaries. 
In  the  body  generally  the  parasites  are  found  in  greatest 
abundance  in  the  spleen,  liver,  and  bone  marrow,  and  also  in 


FIG.  173. — Leishman-Donovan  bodies  from  spleen  smear,      x  1000. 

mesenteric  glands,  especially  in  those  draining  one  of  the 
intestinal  ulcers ;  less  frequently  they  occur  in  the  skin  ulcers, 
and  in  other  parts  of  the  body.  Donovan  described  them  as 
occurring  in  the  peripheral  blood,  especially  within  the  leucocytes, 
and  this  has  been  confirmed  by  other  observers,  though  sometimes 
prolonged  search  is  necessary. 

In  the  body  the  parasite  multiplies  by  simple  fission,  both 
nuclei  dividing  amitotically,  and  two  new  individuals  being 
formed ;  but  sometimes  a  multiple  division  takes  place,  each 
nucleus  dividing  several  times  within  the  protoplasm  and  a 
corresponding  number  of  new  parasites  resulting. 


LEISHMANIA  DONOVANI  633 

In  view  of  Leishman's  original  opinion  an  extremely  important 
discovery  was  made  by  Rogers  and  later  confirmed  by  Leishman 
himself,  to  the  effect  that  in  cultures  a  flagellate  organism 
developed  from  the  Leishman-Donovan  body.  Cultivation  was 
effected  by  taking  spleen  juice  containing  the  parasite,  placing  it  in 
10  per  cent,  sodium  citrate  solution  and  keeping  it  at  17  to  24°  C. 
Under  such  conditions  there  occurs  an  enlargement  of  the 
organism,  but  especially  of  the  larger  nucleus.  This  is  followed 
by  the  appearance  of  a  pink-staining  vacuole  in  the  neighbour- 
hood of  the  smaller  nucleus.  Along  with  these  changes,  in  from 
twenty-four  to  forty-eight  hours  the  parasite  becomes  elongated 
and  the  smaller  nucleus 
and  its  vacuole  move 
to  one  end;  from  the 
vacuole  there  then  ap- 
pears to  develop  a  red- 
staining  tiagellurn,  which 
when  fully  formed  seems 
to  take  its  origin  from 
the  neighbourhood  of 
the  small  nucleus.  The 
body  of  the  parasite  is 
now  from  20  to  22  /x 
long  and  3  to  4  /u.  broad, 
with  the  flagellum  about 
22  fji  long.  The  whole 
development  occupies 
about  ninety-six  hours.  Fl(;  174._Leishmai1-Don0van  bodies  within 
The  formation  of  an  endothelial  cell  in  spleen.  See  also  Plate 
undulating  membrane  VI.,  Fig.  24.  xlOOO. 
\\a-  not  observed,  and, 

although  the  flagellated  organism  moved  flagellum  first,  like  a 
trypanosome,  it  is  evident  that  here  the  relationship  of  the 
micronucleus  is  different,  as  this  structure  lies  anterior  to  the 
macronucleus.  In  his  cultures,  which  kept  alive  for  four  weeks, 
Leishman  made  a  still  further  important  observation.  In  certain 
of  the  flagellate  forms  he  saw  chromatin  granules  develop  in  the 
protoplasm  often  in  couples,  a  larger  and  a  smaller.  There  then 
occurred  a  very  unequal  longitudinal  division  of  the  protoplasm, 
and  a  hair-like  undulating  individual  containing  one  of  the 
pairs  of  chromatin  granules  would  l>e  split  off.  At  first  these 
would  be  non-flagellate,  but  later  a  red-staining  flagellum  would 
appear  at  one  end.  The  analogies  between  these  observations 
and  tho-r  of  Schaudinn  (>'.  p.  GIG)  on  the  relations  of  spirochsetes 


634  LEISHMANIOSIS 

to  trypanosomes  will  be  at  once  apparent ;  the  further  develop- 
ment of  these  spirillary  forms  in  Leishman's  organism  could  not, 
however,  be  traced. 

The  facts  just  detailed  have  been  the  basis  for  discussion  of  the 
classification  of  the  organism,  which  now  usually  goes  by  the 
name  Leishmania  donovani,  originally  given  to  it  by  Ross. 
According  to  one  view,  it  is  to  be  looked  on  as  a  trypanosome, 
and  although,  as  we  have  noted,  its  flagellated  form  differs  from 
the  typical  trypanosoma  form,  it  bears  considerable  resemblance 
to  the  members  of  this  group,  and,  as  Leishman  has  pointed  out, 
his  cultures  may  not  represent  the  full  development  of  the 
organism  in  the  trypanosoma  direction.  Others  have  looked  on 
it  as  a  piroplasma,  but  Minchin's  suggestion  has  been  accepted 
that  in  the  present  incomplete  state  of  knowledge  it  is  well  to 
place  it  and  its  congeners  in  a  provisional  genus,  Leishmania,  of 
the  flagellata. 

The  question  arises,  given  that  the  Leishmania  donovani  is 
the  cause  of  kala-azar,  how  is  infection  spread  ^  On  this  we 
have  as  yet  no  certain  information.  The  fact  that  in  some 
centres  of  the  disease  natives  who  are  supplied  with  good  water 
are  less  liable  than  those  who  rely  on  the  ordinary  polluted 
native  cisterns,  has  led  to  the  opinion  that  water  may  be  the 
carrier  of  infection.  On  the  other  hand,  the  possible  relation- 
ship of  the  organism  to  the  trypanosomata  naturally  suggests 
the  idea  of  an  insect  as  an  intermediary,  and  Rogers  adduced 
some  evidence  that  the  bed-bug  is  the  extra-human  host.  This 
view  was  elaborated  by  Patton,  who  brought  forward  facts  to 
show  that  multiple  cases  might  occur  in  a  house  while 
neighbouring  houses  were  free  from  the  disease.  This  observer 
also  fed  the  common  insect  parasites  of  man  in  Madras  on 
patients  whose  peripheral  blood  contained  the  Leishmania,  and 
found  that  the  parasite  could  be  observed  only  in  the  pediculus 
capitis,  and  in  the  bug,  cimex  macrocephalus.  In  the  midgut  of 
the  latter,  forms  similar  to  those  seen  in  the  earlier  stages  of 
cultures  could  be  found.  Patton  compares  the  organism  to  an 
allied  protozoon  occurring  in  the  intestine  of  the  common  fly. 
The  rarity  of  the  Leishmania  in  the  peripheral  blood  has  been 
advanced  as  an  argument  against  infection  taking  place  by  means 
of  a  blood-sucking  insect,  but  it  has  been  pointed  out  that 
invisible  spirillary  forms  may  be  instruments  of  infection.  It 
may  be  said  here  that  all  attempts  to  communicate  the  disease 
to  animals  have  been  hitherto  unsuccessful. 

With  regard  to  kala-azar  as  a  whole,  we  may  say  that  we  are 
dealing  with  a  distinct  disease  fairly  widespread  in  various  sub- 


LEISHMANIA  INFANTUM  G3.r> 

tropical  regions.  All  attempts  to  include  it  among  the  malarial 
cachexias,  which  clinically  it  so  much  resembles,  have  failed. 
In  this  atypical  cachexial  fever  there  is  always  present  a  parasite 
of  very  special  characters  belonging  or  closely  allied  to  a  group 
which  contains  many  varieties  capable  of  giving  rise  to  similar 
diseases.  Beyond  this  we  cannot  go,  but  at  present  we  must 
admit  that  there  is  strong  presumptive  evidence  of  the  parasite 
described  being  the  cause  of  the  disease. 

Methods  of  Examination. — The  Leishmania  douovani  can  be 
readily  seen  in  films  or  sections  of  the  organs  in  which  we  have 
mentioned  its  occurrence.  These  should  be  stained  by  the 
liomanowsky  stains.  Fluid  taken  from  the  enlarged  spleen  with 
a  perfectly  dry  needle  during  life  may  be  examined,  but  it  is 
probable  that  in  this  disease  puncture  of  the  spleen  may  not  be 
a  very  safe  operation,  as  death  from  haemorrhage  from  this 
organ  is  a  not  uncommon  natural  terminal  event.  During  life  . 
the  main  points  on  which  a  pathological  diagnosis  may  be  based 
a  iv  the  demonstration  of  the  parasite  in  the  circulating  blood 
which  should  always  be  attempted,  the  absence  of  the  malarial 
parasites  from  the  blood,  and  the  features  of  the  leucopenia 
which  have  been  alluded  to. 

Leishmania  Infantum. — Nicolle,  working  in  Tunis,  observed 
a  disease  clinically  identical  with  kala-azar,  but  presenting  the 
peculiarity  of  only  affecting  children  of  about  two  years  of  age. 
Mr  found  in  the  spleen  in  such  cases  an  organism,  microscopically 
indistinguishable  from  the  Leishmania  donovani.  It  was 
cultivated  on  a  modified  Novy  and  MacNeal's  medium,  the 
cultures  presenting  characters  similar  to  those  observed  by 
Rogers  and  by  Leishman  in  the  other  Leishmania.  It  was 
found  that  dogs  could  be  infected  with  the  parasite,  and,  taking 
into  account  the  fact  that  this  animal  is  not  susceptible  to  infec- 
tion with  the  Leishmania  donovani,  and  the  further  fact  that  the 
disease  is  apparently  confined  to  infants,  Nicolle  considered  the 
organism  to  be  a  separate  species  and  gave  it  the  name, 
Leishmania  infantum.  In  his  view,  the  infection  of  the  dog 
possesses  a  further  significance  in  that  this  animal  may  be  the 
reservoir  from  which,  by  means  at  present  unknown,  children 
become  infected.  In  support  of  this,  he  observed  the  fact  that 
a  certain  proportion  of  dogs  destroyed  in  Tunis  contained  the 
parasite  in  the  spleen.  This  disease  occurs  in  other  parts  of  the 
Mediterranean  littoral;  in  1905  Pianese  described  it  in  children 
in  Italy,  and  the  parasites  have  been  found  in  cases  in  that 
country  and  also  in  Sicily  and  Malta. 

Leishmania  Tropica.— In   various   tropical   and  sub-tropical 


636  LEISHMANIOSIS 

regions  (India  and  the  East,  Northern  Africa,  Southern  Russia, 
South  America)  there  is  widely  prevalent  a  variety  of  very 
intractable  chronic  ulceration  which  goes  by  various  names  in 
different  parts  of  the  world — Delhi  sore,  tropical  ulcer,  Aleppo 
boil,  etc.  Various  views  were  formerly  held  as  to  the  pathology 
of  the  condition,  but  the  work  of  J.  H.  Wright  makes  it 
practically  certain  that  a  protozoal  parasite  is  concerned  in  its 
etiology.  In  the  discharge  from  the  ulcer  and  in  sections  of  a 
portion  of  tissue  excised  from  a  case  coming  from  Armenia, 
Wright  observed  great  numbers  of  round  or  oval,  sharply  denned 
bodies,  2  to  4  /x  in  diameter.  When  stained  by  a  Romanowsky 
combination  there  was  found  to  be  a  peripheral  portion  coloured 
a  pale  blue  and  a  central  portion  tending  to  be  unstained ;  there 
were  also  two  chromatin  bodies,  one  larger,  occupying  a  fourth 
or  a  third  of  the  whole  and  situated  in  the  periphery,  another 
smaller,  round  or  rod-shaped,  and  of  a  deeper  colour  than  the 
larger  mass.  It  was  found  that  the  bodies  were  usually  intra- 
cellular  in  position  in  the  lesion,  as  many  as  twenty  being  in  one 
cell,  and  that  the  type  of  cell  containing  them  was,  as  in  kala- 
azar,  that  derivable  from  endothelial  tissues. 

Wright's  observations  have  been .  fully  confirmed  by  workers 
in  various  parts  of  the  world,  and  it  is  now  recognised  that  in 
these  tropical  ulcers  we  have  a  third  example  of  the  activity  of 
a  Leishmania.  This  is  corroborated  by  the  work  of  Row,  who 
has  obtained  cultures  in  citrated  blood,  corresponding  to  those 
of  the  other  two  species.  Nicolle  and  Manceaux  have  also 
cultivated  the  organism  on  Novy  and  MacNeal's  medium,  and 
have  reproduced  the  condition  in  man,  the  monkey,  and  the  dog, 
both  by  virus  obtained  from  the  natural  infection  and  from 
cultures.  The  lesions  were  identical  with  those  naturally  occur- 
ring, but  the  incubation  period  was  often  many  months.  It 
may  be  said  that  Thompson  and  Balfour  have  described  in  the 
Soudan  a  condition  in  which  subcutaneous  nodules  without 
ulceration  occurred  in  man  and  these  contained  Leishmania 
bodies. 

At  present  the  tendency  is  to  look  upon  the  three  Leishmanise 
as  representing  different  species,  but  further  investigation  is  here 
necessary.  It  has  been  pointed  out  that  in  kala-azar,  skin 
ulcerations  occur  which  might  link  this  condition  with  tropical 
ulcer,  but  it  is  to  be  noted  that,  while  in  the  latter  enormous 
numbers  of  the  parasite  are  found,  in  the  ulcers  of  kala-azar,  on 
the  other  hand,  parasites  are  difficult  to  find.  Again,  Nicolle 
has  found  that  dogs  infected  with  Leishmania  tropica  appeared 
to  be  not  so  susceptible  to  subsequent  infection  with  Leishmania 


PIROPLASMOSIS  637 

infantum.     These  facts,  however,  might  be  consistent  with  the 
existence  of  three  species. 

Histoplasma  Capsulatum. — Under  this  name,  Darling  has  described  a 
parasite  observed  by  him  in  Panama,  in  certain  cases  characterised 
during  life  by  continued  irregular  fever,  spleno-megaly,  emaciation,  and 
ana-mia,  and  post  mortem  showing  minute  granulomata  in  the  lungs, 
irregular  necrosis  and  cirrhosis  of  the  liver, — the  spleen,  naked-eye, 
resembling  that  of  spleno-mvelogenous  leukemia.  In  smears  from  the 
lung  nodules,  the  liver,  and  spleen,  stained  by  Irishman's  method, 
there  were  observed  enormous  numbers  of  small  bodies  sometimes  crowd- 
ing endothelial  cells,  often  free.  These  bodies  were  round  or  oval  and 
from  1  to  4  /x  in  diameter.  Each  contained  an  irregularly  placed 
chromatin  mass,  the  shape  of  which  was  globular,  oval  or  kidney-shaped, 
the  remainder  of  the  parasite  consisting  of  bine-staining  basophilic 
substance.  The  parasite  is  surrounded  by  a  non-staining  refractile 
capsule,  one-sixth  of  the  diameter  of  the  parasite  in  width  and  sometimes 
containing  a  single  minute  chromatoid  dot,  and  similar  granules  are 
sometimes  seen  in  the  non-chromatoid  part  of  the  body  of  the  parasite. 
Darling  considers  this  organism  to  be  different  from  the  Leishmania 
donovani  in  the  form  and  arrangement  of  its  chromatin  and  in  not 
possessing  a  blepharoplast. 

PlKOI'LASMOSIS. 

Up  to  the  present  no  human  disease  has  been  proved  to  be  associated 
with  the  presence  of  piroplasmata.  The  observations  of  Donovan, 
which  seemed  to  indicate  that  the  parasite  of  kala-azar  might  be  found 
within  the  red  blood  corpuscles,  and  which  led  Laveran  to  denominate 
the  Leishmania  donovani  the  piroplasma  donovani,  have,  as  already 
indicated,  not  been  confirmed  ;  the  same  is  true  of  the  association  of 
piroplasms  with  the  occurrence  of  the  Rocky  Mountain  spotted  fever 
sometimes  prevalent  in  Montana.  But  several  important  diseases  of  the 
lower  animals  are  almost  certainly  caused  by  protozoan  parasites  of  this 
group,  and  a  short  account  of  the  organisms  may  be  given. 

The  piroplasmata  are  pear-shaped  unicellular  organisms  about  1  to  1  '5  /* 
long  and  varying  in  breadth.  The  peripheral  part  is  denser  than  the 
central,  whicl'i  often  appears  as  if  vacuolated,  and  at  the  broad  end  there 
is  ;i  well-staining  chromatin  mass.  Sometimes  irregular  and  ring-,  rod-, 
or  oval-shaped  individuals  occur.  The  organisms  are  found  within  the 
red  Mood  corpuscles  of  the  infected  animal  and  also  free  in  the  blood.  In 
the  former  situation  there  is  sometimes  only  one  within  a  cell,  but  the 
numbers  vary  under  different  circumstances  and  in  different  species. 
Multiplication  takes  place  by  fission,  and  the  new  individuals,  remaining 
fui  longer  or  shorter  times  in  apposition,  account  for  some  of  the  appear- 
•noea  seen  in  cells.  Especially  in  the  forms  free  in  the  blood  pseudopodial 
prolongations  of  the  protoplasm,  usually  from  the.  pointed  end,  arc 
developed,  and  it  may  be  by  means  of  such  pseudopodia  that  entrance  to 
the  red  cells  is  obtained.  Infection  fs  usually  carried  from  infected 
unimals  by  means'of  ticks.  In  one  case  Koch  has  described  the  develop- 
ment in  the  organism,  in  the  stomach  of  the  tick,  of  spiked  protoplasmic 
processes  sprouting  out  from  the  broad  end  of  the  piro plasm,  and  the 
occurrence  of  conjugation  of  two  such  individuals  by  their  narrow  ends 
to  form  a  xygote.  Further  observations,  however,  here  are  necessary, 


638  PIROPLASMOSIS 

and  nothing  is  known  of  the  further  history  of  the  parasite  within  the 
insect  except  that  the  eggs  in  the  ovary  may  become  infected,  so  that 
insects  developed  from  these  can  carry  infection  to  animals.  Frequently 
when  an  animal  has  passed  through  an  attack  of  a  piroplasmosis  it  is 
immune  to  the  disease,  and  with  regard  to  this  immunity  in  certain  cases 
very  interesting  facts  have  been  observed.  For  instance,  the  condition 
may  not  be  associated  with  the  disappearance  of  the  parasite  from  the 
blood  of  the  immune  animal,  and  the  latter  may  thus  be  a  source  of 
danger  to  other  non-immune  animals  with  which  ticks  harboured  by  it 
may  come  in  contact. 

The  following  are  the  chief  piroplasmata  causing  disease  in  animals  : — 
(i)  Piroplasma  bigeminum.  This  was  first  described  by  Theobald  Smith 
and  is  the  cause  of  Texas  or  red- water  fever,  a  febrile  condition  associated 
with  hffimoglobinuria,  which  occurs  in  the  Southern  States  of  America, 
the  Argentine,  South  and  Central  Africa,  Algeria,  various  parts  of 
Northern  Europe,  and  in  Australia.  The  organism  gets  its  name  of 
bigeminum  from  the  fact  that  it  is  often  present  in  the  red  cells  in  pairs, 
which  may  be  attached  to  one  another  by  a  fine  thread  of  protoplasm  ; 
this  probably  results  from  the  complete  separation  of  two  individuals 
being  delayed  after  division  has  occurred.  Infection  is  here  spread  by 
the  tick  boophilus  bovis,  and  some  of  the  characteristics  of  the  disease 
epidemiologically  are  explained  by  the  fact  that  this  insect  goes  through 
all  its  moultings  on  the  same  individual  host.  (2)  Piroplasma parvum. 
This  organism  was  discovered  by  Theiler  in  the  blood  of  cattle  suffering 
from  African  East  Coast  fever,  a  disease  closely  resembling  Texas  fever, 
which  prevails  endemically  in  a  narrow  strip  along  a  long  extent  of  the 
east  coast,  and  which  occurs  epidemically  inland.  As  its  designation 
implies,  the  organism  is  small,  and  it  is  also  attenuated.  Its  insect  host 
is  the  tick  rhipicephalus  appendiculatus,  and  it  may  be  noted  that  this 
tick  drops  off  the  animal  on  which  it  may  be  feeding  when  it  is  about  to 
go  through  one  of  its  several  moultings.  It  can  thus  carry  an  infection 
much  more  quickly  and  widely  through  a  herd  than  can  the  carrier  of 
ordinary  red-water  fever.  It  may  be  said  that  in  England  there  occurs  a 
red-water  fever  also  associated  with  the  presence  of  a  piroplasm  in  the 
blood,  but  the  relationship  of  this  organism  to  the  other  varieties  has  not 
yet  been  fully  worked  out.  (3)  Piroplasma  equi.  This  organism  gives 
rise  to  biliary  fever  in  horses,  another  South  African  disease,  and  it  is 
carried  by  the  tick  rhipicephalus  evertsii.  In  this  disease  Theiler  made 
the  interesting  observation  that  when  the  blood  of  a  donkey  which  had 
recovered  from  the  disease  was  injected  into  a  horse,  the  latter  suffered  a 
slight  illness  only,  although  the  organisms  were  present  in  the  blood 
injected.  Such  a  fact  is  of  importance,  as  attenuation  of  virulence  in 
pathogenic  protozoa  seems,  so  far  as  our  present  knowledge  goes,  a  not 
very  common  event.  (4)  Piroplasma  canis.  This  causes  a  piroplas- 
mosis occurring  in  dogs. 

With  regard  to  the  pathology  of  infection  by  piroplasmata  we  know 
nothing.  The  diseases  are  often  extremely  fatal,  carrying  off  nearly 
every  individual  attacked,  but  we  do  not  know  the  nature  of  the  changes 
originated. 


APPENDIX  F. 

YELLOW   FEVER. 

YKLLOW  fever  is  an  infectious  disease  which  is  endemic  in  the 
\\  Yst  Indies,  in  Brazil,  in  Sierra  Leone  and  the  adjacent  parts 
of  West  Africa,  though  it  is  probable  that  it  was  from  the 
first-named  region  that  the  others  were  originally  infected.  . 
I  Y<>m  time  to  time  serious  outbreaks  occur,  during  which 
neighbouring  countries  also  suffer,  and  the  disease  may  be 
carried  to  other  parts  of  the  world.  In  this  way  epidemics 
have  occurred  in  the  United  States  and  elsewhere,  infection 
usually  being  carried  by  cases  occurring  among  the  crews 
of  ships.  In  the  parts  where  it  is  endemic,  though  usually 
a  few  cases  may  occur  from  time  to  time,  there  is  some 
evidence  that  occasionally  the  disease  may  remain  in  abeyance 
for  many  years  and  then  originate  de  novo.  There  is,  there- 
t'mv,  reason  to  suspect  that  the  infective  agent  can  exist  for 
considerable  periods  outside  the  human  body.  It  is  possible, 
however,  that  continuity  may  be  maintained  by  the  occurrence 
of  a  mild  type  of  the  disease,  which  may  be  grouped  with  the 
"  bilious  fevers  "  prevalent  in  yellow  fever  regions.  This  would 
explain  the  degree  of  immunity  which  is  shown  during  a  serious 
epidemic  by  the  older  immigrants. 

Great  variations  are  observed  in  the  clinical  types  under 
which  the  disease  presents  itself.  Usually  after  from  two  to 
six  days'  incubation  a  sudden  onset  in  the  form  of  a  rigor 
occurs.  The  temperature  rises  to  104-105°  F.  The  person  is 
livid,  with  outstanding  bloodshot  eyes.  There  are  present  great 
prostration,  pain  in  the  back,  and  vomiting,  at  first  of  mucus, 
later  of  bile.  The  urine  is  diminished  and  contains  albumin. 
About  the  fifth  day  an  apparent  improvement  takes  place,  and 
this  may  lead  on  to  recovery.  Frequently,  however,  the  remission, 
which  may  last  from  a  few  hours  to  two  days,  is  followed 
by  an  aggravation  of  all  the  symptoms.  The  temperature  rises, 
jaundice  is  observed,  haemorrhages  occur  from  all  the  mucous 

63» 


640  YELLOW  FEVER 

surfaces,  causing,  in  the  case  of  the  stomach,  the  "  black  vomit " 
— one  of  the  clinical  signs  of  the  disease  in  its  worst  form. 
Anuria,  coma,  and  cardiac  collapse  usher  in  a  fatal  issue.  The 
mortality  varies  in  different  epidemics  from  about  35  to  99 
per  cent,  of  those  attacked.  Both  white  and  black  races  are 
susceptible,  but  those  who  have  resided  long  in  a  country  are 
less  susceptible  than  newr  immigrants.  An  attack  of  the  disease 
usually  confers  complete  immunity  against  subsequent  infection. 

Post-mortem  the  stomach  is  found  in  a  state  of  acute  gastritis, 
and  contains  much  altered  blood  derived  from  haemorrhages 
which  have  occurred  in  the  mucous  and  submucous  coats.  The 
intestine  may  be  normal,  but  is  often  congested  and  may  be 
ulcerated  ;  the  mesenteric  glands  are  enlarged.  The  liver  is  in 
a  state  of  fatty  degeneration  of  greater  or  less  degree,  but  often 
resembling  the  condition  found  in  phosphorus  poisoning.  The 
kidneys  are  in  a  state  of  intense  glomerulo-nephritis,  with  fatty 
degeneration  of  the  epithelium.  There  is  congestion  of  the 
meninges,  especially  in  the  lumbar  region,  and  haemorrhages 
may  occur.  The  other  organs  do  not  show  much  change, 
though  small  haemorrhages  under  the  skin  and  into  all  the 
tissues  of  the  body  are  not  infrequent.  In  the  blood  a  feature 
is  the  excess  of  urea  present,  amounting,  it  may  be,  to  nearly 
4  per  cent. 

Etiology  of  Yellow  Fever. — Although  a  large  amount  of 
bacteriological  work  has  been  done  on  yellow  fever,  this  has 
merely  a  historical  interest,  as  it  is  now  known  that  the 
causal  agent  is  not  one  of  the  ordinary  bacteria,  but  belongs  to 
the  group  of  ultra-microscopic  organisms.1  A  mosquito  acts  as 
the  intermediate  host,  and  the  facts  detailed  below  point  to  the 
organism  passing  through  some  cycle  of  development  in  the 
body  of  the  insect.  The  analogy  of  malaria  makes  it  extremely 
probable  that  the  organism  is  a  protozoon,  but  this  has  not  yet 
been  completely  proved.  As  bacteriological  work  led  up  to  the 
establishment  of  our  knowledge  regarding  the  nature  of  the 
disease,  some  reference  must  be  made  to  it. 

A  very  full  research  into  the  bacteriology  of  yellow  fever  was- 
that  of  Sternberg,  and  one  of  the  organisms  isolated,  which  he 
called  the  bacillus  x,  appeared  possibly  to  have  some  relationship 
to  the  disease.  Sanarelli  in  1897  isolated  an  organism  Avhich  he 
called  bacillus  icteroides,  and  which  he  considered  to  be  the 
cause  of  yellow  fever  ;  it  was  probably  identical  with  the  bacillus 

1  In  several  diseases  the  existence  of  such  causal  factors  is  probable. 
Examples  in  animals  are  foot  and  mouth  disease,  South  African  horse  sickness, 
and  the  contagious  pleuro-pueumonia  of  cattle. 


ETIOLOGY  OF  YELLOW  FEVER  641 

x  of  Sternberg,  but  subsequent  observations  made  by  others 
gave  conflicting  results.  The  bacillus  icteroides,  as  described  by 
Sanarelli,  belongs  to  the  paratyphoid  group,  possessing  lateral 
fiagella,  growing  on  gelatin  without  liquefaction,  and  fermenting 
glucose  but  not  lactose.  Reed  and  Carroll  found  that  it  was 
practically  identical  with  the  bacillus  of  swine  cholera.  It 
must  now  be  considered  merely  as  an  organism  which  may 
occur  in  the  organs  and  tissues  in  yellow  fever  as  a  secondary 
infection,  but  without  any  etiological  significance. 

The  facts  of  importance  which  have  been  established 
regarding  the  etiology  of  the  disease  are  due  to  the  labours  of 
the  United  States  Army  Commission,  which  began  its  wrork  in 
1900.  The  members  of  the  Commission  first  directed  their 
inquiries  towards  determining  whether  the  bacillus  icteroides 
was  present  in  the  blood  during  life,  and  a  series  of  cases  was 
investigated  bacteriologically,  with  entirely  negative  results  in 
each  instance.  They  then  resolved  to  test  the  hypothesis  of 
Dr.  Carlos  Finlay  of  Havana,  promulgated  several  years  pre- 
viously, that  the  disease  wras  carried  by  mosquitoes.  Selecting 
mosquitoes  which  they  reared  from  eggs,  they  allowed  them  to 
bite  yellow  fever  patients  and  then  to  bite  healthy  men.  Of 
several  experiments  of  this  nature  two  were  successful  in  the 
first  instance,  the  first  individual  to  be  infected  in  this  way 
being  Dr.  James  Carroll,  a  member  of  the  Commission,  who 
passed  through  a  severe  attack  of  typical  yellow  fever.  Experi- 
ments were  then  performed  on  a  larger  scale,  with  completely 
confirmatory  results,  as  to  the  conveyance  of  the  disease  by 
mosquitoes.  Of  twelve  non-immunes  living  under  circumstances 
which  excluded  natural  means  of  infection,  ten  contracted 
yellow  fever  after  hiving  been  bitten  by  mosquitoes  which  had 
previously  bitten  yellow  fever  patients ;  happily  all  of  these 
recovered.  Two  of  the  men  who  were  thus  infected  had  been 
previously  exposed  to  contact  with  fomites  from  yellow  fever 
patients  without  results.  These  results  were  confirmed  by 
(luiu'-ras,  whose  investigations  were  carried  out  along  similar 
lines  ;  of  seventeen  individuals  bitten  by  infected  mosquitoes, 
eight  took  yellow  fever,  and  three  of  these  died. 

The  species  of  mosquito  used  by  the  American  Commission 
was  the  Stefjomyia  fasciata,  and  up  to  the  present  time  no  other 
s]iecies  has  been  found  capable  of  carrying  the  infection.  It  has 
also  been  determined  that  a  certain  }>eriod  must  elapse  after  the 
insect  has  bitten  a  yellow  fever  patient  before  it  becomes  infec- 
tive to  another  subject.  In  summer  weather  this  period  is  about 
twelve  days  ;  at  a  lower  temperature  somewhat  longer.  This 


642  YELLOW  FEVER 

probably  means  that,  as  in  the  case  of  malaria,  the  parasite  must 
pass  through  certain-  stages  of  development  before  it  reaches  the 
salivary  gland  and  is  thus  in  a  position  to  be  transferred  to  a  fresh 
subject.  Infected  mosquitoes,  however,  retain  the  power  of 
infection  for  a  considerable  time  afterwards,  probably  as  long  as 
sixty  days.  It  has  also  been  shown  that  mosquitoes  may  become 
infective  after  biting  a  patient  on  the  first,  second,  or  third  day 
of  the  disease,  but  at  a  later  period  the  results  are  usually 
negative,  apparently  because  the  virus  is  no  longer  present  in 
the  blood. 

Interesting  results  were  also  obtained  with  regard  to  the 
communication  of  the  disease  directly  from  patient  to  patient, 
the  conclusion  arrived  at,  after  careful  experiments,  being  that 
the  disease  cannot  be  transferred  in  this  way,  even  when  the 
contact  is  of  a  close  character.  In  a  specially  constructed  house 
seven  men  were  exposed  to  the  most  intimate  contact  with  the 
fomites  of  yellow  fever  patients  for  a  period  of  twenty  days  each, 
the  soiled  garments  worn  by  the  patients  being  in  some  cases 
actually  slept  in  by  these  men  ;  the  result  was  that  not  one  of 
those  thus  exposed  contracted  the  disease.  The  conclusions  on 
this  point  have  been  subsequently  confirmed  by  other  workers. 

The  American  Commission  also  found  it  possible  to  transmit 
yellow  fever  to  a  healthy  man  by  injecting  small  quantities  of 
blood  or  of  serum  taken  from  a  yellow  fever  patient  at  any 
period  up  till  the  third  day  of  the  disease.  The  period  of 
incubation  in  this  case  is  somewhat  shorter  than  when  the  disease 
is  conveyed  by  the  bite  of  mosquitoes,  the  average  duration  in 
the  former  case  being  about  three  days,  and  in  the  latter  about 
four  days,  though  these  times  may  be  considerably  exceeded. 
It  is  also  interesting  to  know  that  in  these  experimental  injec- 
tions the  blood  or  serum  used  was  found  to  be  free  from  bacteria. 
Up  till  the  present  time,  we  know  of  only  these  two  methods  of 
infection,  namely,  indirectly  by  the  bite  of  a  mosquito  infected 
with  the  yellow  fever  germ;  or  directly  by  the  injection  of  some 
of  the  blood  from  a  yellow  fever  patient.  In  these  respects 
there  is  a  striking  similarity  to  what  has  been  established  in  the 
case  of  malarial  fever. 

Experiments  with  regard  to  the  nature  of  the  yellow  fever 
organism  were  carried  out  by  Reed  and  Carroll,  and  interesting 
results  were  obtained.  They  found  that  the  organism  of  the 
disease  was  very  easily  killed  by  heat,  as  blood  from  a  yellow 
fever  patient  lost  its  infective  power  on  being  heated  to  55°  C. 
for  ten  minutes.  On  the  other  hand,  blood  or  serum  was  found 
to  be  still  infective  after  having  been  passed  through  a  Berkefeld 


ETIOLOGY  OF  YELLOW  FEVER  643 

tilt.-].  This  has  been  confirmed  by  the  French  Commission, 
with  tin-  additional  result  that  the  virus  passes  through  a 
( 'liaiiil>er]and  F  filter,  but  not  through  a  Chamberland  B.  These 
facts  would  show  that  the  parasite  is  of  extremely  minute  size, 
and  apparently  belongs  to  the  group  of  ultra-microscopic 
organisms.  l.Tp  till  the  present  time  all  attempts  to  find  by 
microscopic  examination  the  yellow  fever  parasite,  either  in  the 
blood  of  patients  suffering  from  the  disease  or  in  the  tissues  of 
infective  mosquitoes,  have  been  attended  with  negative  results. 
It  has  U vii  recently  stated  that  it  is  possible  to  produce  yellow 
fever  in  the  chimpanzee  by  the  injection  of  blood  from  a  patient. 
Though  nothing  has  been  determined  regarding  the  actual 
nature  of  the  virus,  yet  the  results  already  obtained  have 
supplied  the  basis  for  preventive  measures  against  the  disease, 
these  being  directed  towards  the  destruction  of  mosquitoes  and 
the  protection  of  those  suffering  from  yellow  fever,  and  also  the 
healthy,  against  the  bites  of  these  insects.  Already  a  striking 
dfgivc  of  success  has  been  obtained  in  Havana.  Such  measures 
came  into  force  in  February  1901,  and  in  ninety  days  the  town 
was  free  of  yellow  fever,  and  for  fifty-four  days  later  no  new 
cased  occurred;  and  although  subsequently  the  disease  was 
reintroduced  into  the  town,  no  difficulty  was  experienced  in 
stamping  it  out  by  the  same  measures.  In  recent  years  the 
results  have  also  been  highly  gratifying,  and  the  disease  may 
be  said  to  be  practically  eradicated  from  Havana.  In  other 
places  also  successful  results  have  been  obtained,  and  epidemics 
would  ap]n'«ir  to  be  now  under  control  if  the  proper  measures 
are  taken. 


APPENDIX   G. 

EPIDEMIC  POLIOMYELITIS. 

DURING  the  past  twenty  years,  the  occurrence  at  one  time  and 
place  of  groups  of  cases  of  acute  anterior  poliomyelitis  has  favoured 
the  idea  that  the  condition  might  be  of  an  infective  nature. 
This  view  was  supported  by  the  work  of  Landsteiner  and  Popper, 
who,  in  1909  in  Vienna,  succeeded  in  producing  the  disease  in  a 
monkey  by  the  intraperitoneal  injection  of  an  emulsion  of  the  spinal 
cord  of  a  child  who  had  succumbed  on  the  fourth  day  of  illness. 
The  occurrence  in  New  York  in  the  summer  of  1907  of  an 
epidemic  in  which  probably  over  2000  cases  occurred,  762 
of  which  were  carefully  investigated  by  a  Commission, 
concentrated  attention  in  America  upon  the  condition,  and  a 
recrudescence  in  the  summer  of  1909  furnished  Flexner  with 
material  for  investigation.  In  earlier  experiments  it  had  been 
observed  that  the  cerebro-spinal  fluid  was  non-infective,  and 
that,  while  it  was  possible  to  infect  monkeys  with  the  disease, 
the  transference  of  the  condition  from  monkey  to  monkey  wTas 
not  successful.  Flexner  found  that  if  for  intraperitoneal 
injection  intracerebral  inoculation  was  substituted,  the  brain  and 
cord  of  the  infected  animals  was  infective  for  other  monkeys, 
the  incubation  period  being  from  four  to  thirty-three  days.  In 
this  way  the  disease  was  kept  up  by  subdural  and  intravenous 
injection,  and  also  by  injection  into  the  sheath  of  nerves  such  as 
the  sciatic, — the  intraperitoneal  and  subcutaneous  methods  being 
found  also  to  give  results.  The  disease  produced  resembled  in 
every  way  the  disease  naturally  occurring  in  children,  and 
frequently  resulted  fatally,  as  in  the  natural  illness.  When 
the  virus  was  injected  into  the  sheath  of  a  nerve,  the  paralytic 
symptoms  first  appeared  in  relation  to  that  part  of  the  cord 
from  which  the  nerve  emerges.  Infective  material  preserved  in 
glycerin  retained  its  virulence, — a  fact  which  rather  militates 
against  there  being  a  bacterial  virus, — and  the  virus  could  also 
withstand  prolonged  freezing  and  drying  for  a  considerable  period. 

644 


EPIDEMIC  POLIOMYELITIS  645 

In  animals  other  than  monkeys  no  result  followed  infection.  A 
raivful  microscopic  examination  of  the  nervous  system  in  natural 
and  experimental  cases  did  not  disclose  the  presence  of  bacteria 
or  protozoa,  and  it  was  found  that  the  virus  could  pass  through 
a  llrrkefeld  lilter  without  losing  its  infectivity,  and  that  the  cords 
of  animals  thus  infected  were  still  infective  for  further  animals, — 
men-  toxic  action  being  thus  excluded.  These  results  have  been 
continued  by  Levaditi  working  with  the  chimpanzee, and  then-  is 
thus  little  doubt  that  here  again  we  are  dealing  with  an  ultra- 
micruscnj.ic  vims.  Flexner  found  that  when  the  virus  was 
mixed  with  bouillon  a  turbidity  developed,  but  no  formed 
organic  body  could  be  detected.  He  further  found  that  monkeys 
which  had  passed  through  the  illness  following  inoculation  \\ere 
insusceptible  to  re-inoculation,  and  both  he  and  Levaditi  noted 
that  the  serum  of  such  insusceptible,  monkeys  had  the  capacity 
of  neutralising  the  virus,  a  similar  result  being  obtained  with 
the  serum  of  human  cases  which  had  recovered. 

With  regard  to  the  distribution  of  the  virus  in  an  infected 
animal,  the  chief  concentration  is  found  in  the  nervous  system, 
but  it  was  also  found  in  lymphatic  glands  ;  as  has  been  stated, 
the  cerebro-spinal  fluid  is  apparently  inert.  No  facts  are  known 
bearing  on  the  channel  of  natural  infection  or  on  the  path  by 
which  the  poison  reaches  the  cord, — whether  by  the  general 
lymph  stream  or  by  the  sheaths  of  nerves,  but  the  virus  can 
be  eliminated  by  the  nose, 'and  infection  can  also  be  effected 
through  the  scarified  nasal  mucosa.  In  its  pathology  the 
condition  bears  many  resemblances  to  rabies. 

These  recent  observations  are  of  the  greatest  importance  in 
relation  to  the  etiology  of  the  condition  and  also  possibly  in 
relation  to  treatment. 


APPENDIX  H. 

PHLEBOTOMUS  FEVER. 

IN  Dalmatia,  Herzegovina,  and  neighbouring  parts  of  the 
Adriatic  littoral  there  occurs  a  disease  known  as  "pappataci," 
characterised  by  fever  lasting  for  about  three  clays,  followed 
by  somewhat  prolonged  prostration,  but  very  rarely  having  a 
fatal  issue.  Doerr,  after  failing  to  isolate  any  organism  from 
the  blood,  found  that  the  subcutaneous  injection  of  from  '5  to 
1  c.c.  of  the  serum  from  a  case  into  a  healthy  individual  was 
followed  about  eight  days  later  by  an  attack  of  the  disease. 
A  similar  effect  was  produced  with  the  serum  after  it  had  been 
passed  through  a  Berkefeld  filter, — all  the  inoculation  experiments 
being  performed  at  a  distance  from  the  original  location  of  the 
disease.  The  view  is  therefore  put  forward  that  here  we  have 
to  deal  with  another  example  of  an  ultra-microscopic  virus.  The 
disease  has  been  only  observed  in  the  summer  season,  and  Doerr 
considered  there  was  justification  for  the  popular  view  that 
it  was  associated  with  the  bite  of  the  dipterous  fly,  phlebotomm 
pappatasii.  This  was  borne  out  by  the  fact  that  on  feeding 
such  flies  on  a  sick  person,  transporting  them  to  a  locality  free 
from  the  disease  and  allowing  them  to  bite  healthy  individuals, 
the  affection  was  reproduced.  An  apparently  identical  disease 
occurs  in  Malta  and  has  been  investigated  by  Birt  under  the 
name  of  "Phlebotomus  Fever."  This  observer  fully  confirmed 
Doerr's  results,  the  condition  again  being  reproduced  by  infected 
flies  which,  however,  were  found  not  to  manifest  infectivity 
earlier  than  seven  days  after  biting. 

These  results  are  of  importance  of  themselves  as  throwing 
light  on  the  etiology  of  a  troublesome  disease  of  the  Mediter- 
ranean littoral,  but  they  are  also  interesting  as  having  a  possible 
bearing  on  the  pathology  of  a  group  of  similar  affections 
occurring  in  various  parts  of  the  world, — chiefly  in  coastal  areas, 
— and  going  under  a  variety  of  names.  Examples  are  dengue, 
the  three-day  fever  of  various  regions,  Canary  fever,  Shanghai 

C46 


PHLEBOTOMUS  FEVER  647 

fever,  Chitral  fever,  and  the  seven-day  fever  or  simple  continued 
fever  of  India.  Of  these,  that  presenting  the  most  definite 
clinical  picture  is  dengue, — a  condition  for  long  well  known  and 
having  an  extensive  distribution,  and  it  may  be  said  that 
Ashburn  and  Craig  in  the  Philippines  found  the  blood  in  dengue 
as  in  pappataci  to  be  infective  even  after  filtration.  Whether 
all  tlie.se  disease  conditions  are  identical  further  research  must 
decide;  at  present  Birt  believes  that  at  any  rate  pappataci 
and  dengue  are  distinct,  and  certainly  Doerr  does  not  in  his 
description  allude  to  the  terminal  skin  eruption  which  Manson 
believes  to  be  of  very  constant  occurrence  in  the  latter.  The 
rarity  of  a  fatal  result  in  these  diseases  makes  their  investiga- 
tion by  inoculation  of  the  human  subject  relatively  safe. 


APPENDIX   J. 

TYPHUS  FEVER. 

UP  till  recently  all  attempts  to  elucidate  the  etiology  of  this  dis- 
ease by  ordinary  bacteriological  methods  have  given  equivocal 
results.  Certain  experiments,  however,  performed  by  Nicolle 
in  1909,  during  an  outbreak  in  Tunis,  are  of  importance.  This 
observer  found  that  the  inoculation  of  the  monkeys,  macacus 
cynomolgus  and  macacus  sinicus,  with  typhus  blood  gave  a 
negative  result.  On  injecting  1  c.c.  of  such  blood  into  a 
chimpanzee,  however,  an  illness  presenting  the  features  of  the 
disease  in  man,  including  the  eruption,  resulted  three  days  later, 
and  death  occurred.  It  was  then  found  that  the  blood  of  this 
animal  was  capable  of  originating  a  similar  disease  after  an 
incubation  period  of  ten  to  fourteen  days  in  the  lower  apes 
referred  to,  and  this  was  kept  up  through  several  passages. 
The  virulence  of  the  material,  however,  gradually  died  out. 
The  dog  and  the  white  rat  were  insusceptible.  It  was  found 
that  macacus  sinicus  could  be  infected  by  means  of  the  body 
louse,  the  incubation  period,  however,  being  lengthened  to 
forty  days.  These  experiments  probably  throw  important  light 
on  the  etiology  of  the  condition,  and  on  the  means  by  which 
the  disease  is  spread  in  man. 


648 


BIBLIOGRAPHY. 


L  TKXT- BOOKS. — In  English  the  student  may  consult  the  follow- 
ing :  "  Micro-organisms  and  Disease,"  E.  Klein,  3rd  ed.,  London,  1896. 
"Bacteriology  and  Infective  Diseases,"  Edgar  M.  Crookshank,  London, 
1898.  "A  Manual  of  Bacteriology,"  George  M.  Sternberg,  New  York, 
1st  ed.  1893,  2nd  ed.  1896  (this  book  contains  a  full  bibliography). 
"Text-Book  upon  the  Pathogenic  Bacteria,"  Joseph  M'Farland,  London, 
f.th  ed.  1906.  "Practical  Bacteriology,"  A.  A.  Kanthack  and  J.  H. 
Diysdalc,  London,  189;").  "  Bacteria  and  their  Products,"  0.  S.  Wood- 
head,  London,  1891.  "  Bacteriological  Technique,"  Eyre,  London,  1902. 
Tin-  articles  on  bacteriological  subjects  in  CliHbnl  Allbutt's  "  System  of 
Medicine,"  London,  1906-10,  are  of  the  highest  excellence,  and  have  full 
bibliographies  appended.  For  the  hygienic  aspects  of  bacteriology,  see 
"  System  of  Hygiene,"  Stevenson  and  Murphy,  London,  1892-94. 

For  non-pathogenic  bacteria  occurring  in  connection  with  pathological 
\\<>rk  consult  Heim,  op.  cit.  infra.  For  fungi,  see  I)e  Bary,  "Comparative 
Morphology  and  Biology  of  the  Fungi,  Mycetoxoa  and  Bacteria/'  transl. 
by  Garnsey  and  Balfour,  Oxford,  1887  ;  Sachs,  "Text-Book  of  Botany," 
ii.,  transl.  by  Garnsey  and  Balfour,  Oxford,  1887. 

In  German:  "Die  Mikroorganismen,"  by  Dr.  C.  Fliigge,  3rd  ed., 
Leipzig,  1896.  "  Lehrbuch  der  pathologischen  Mykologie,"  by  Baum- 
-•••u  ten,  Braunschweig,  1890.  "  Handbuch  der  pathogenen  Mikro- 
ui-gaiiismen,"  Knlle  and  \Vassermann,  Fischer,  Jena,  1904.  "Handbuch 
<1.  i  Tfrluiik  and  Methodikderlmmunitiitsforschung,"  Krausand  Levaditi, 
Jena,  1908  and  1909. 

In  French  :  Roger,  "  Les  maladies  infectieuses,"  Paris,  1902. 

I'KKioiui  ALS.—  For  references  to  current  work  see  (1)  Uentralbl.  f. 
lla.ktcriol.  u.  Parasitenk.,  Jena.  This  publication  commenced  in  1887. 
Two  volumes  are  issued  yearly.  In  1895  it  was  divided  into  two  parts. 
Abtheilung  I.  deals  with  Medizinisch-hygienischc  Bakteriologie  und 
ihi'-rixrhc  I'anisitenkuwle.  The  volumes  of  this  part  are  numbered  cpn- 
st(  utively  with  those  of  the  former  series,  the  first  issued  thus  being 
vol.  xvii.  Commencing  in  1902  with  volume  xxxi.,  each  volume  of 
Abtheilung  I.  was  further  divided  into  two  parts,  one  consisting  of 
Originate,  the  other  of  Jlefcmte.  Abtheilung  II.  deals  with  A //</•;- 
,,i ''iin-  /<iii>lifirlsch(tftli<-)i-t«-liiinlogisclie  Bakterioloyie,  O&rwtgs-^kynoloffif 
nii'l  rjlanzenpatli »/<»//'<'.  The  first  volume  is  entitled  Zweite  Abtheilung, 
1 5-1.  L  It  contains  original  articles,  Referate,  etc.  (2)  Bull.  dcVInst. 
Pasteur,  Paris,  Masson.  Besides  bacteriological  abstracts  this  journal 
contains  many  valuable  reviews  and  analyses  relating  to  protozoology. 

649 


650  BIBLIOGRAPHY 

(3)  "Ergebnisse  der  allgemeinen  Pathologic,"  Lubarsch  and  Ostertag, 
Wiesbaden,  Bergrnann.  This  from  time  to  time  contains  valuable 
critical  reviews. 

The  most  complete  account  of  the  work  of  the  year  is  found  in  the 
Jahresb.  it.  d.  Fortschr.  .  .  .  d.  path.  Mikrooryanismen,  conducted  by 
Baumgarten,  and  published  in  Braunschweig.  This  publication  com- 
menced in  1887.  Its  disadvantage  is  that  the  volume  for  any  year  does 
not  usually  appear  till  two  years  later. 

Bacteriology  is  also  dealt  with  in  the  Index  Medicus.  For  valuable 
lists  of  papers  by  particular  authors  see  Royal  Society  Catalogue  of 
Scientific  Papers  and  Internat.  Cat.  Sc.  Lit.  (Section,  Bacteriology). 

The  chief  bacteriological  periodicals  are  the  Journ.  Path,  and  Bacteriol, , 
Cambridge,  edited  by  G.  Sims  Woodhead  ;  the  Ztschr.  f.  Hyg.  n.  Infec- 
fionskrankh.,  Leipzig,  edited  by  Koch  and  Flligge ;  the  Ztxchr.  f. 
Immunitatsforschuny,  Frankfort,  edited  by  Ehrlich  ;  and  the  Ann.  de 
Vlnst.  Pasteur,  Paris,  edited  by  Duclaux  ;  Journ.  Krper.  Med.,  New 
York,  edited  by  Flexner  ;  Journ.  Hyij.,  Cambridge,  edited  by  Nuttall ; 
Journ.  Med.  Research,  Boston,  edited  by  Ernst ;  Journ.  Infect.  Disease*, 
Chicago,  edited  by  Hektoen  ;  Journ.  of  Royal  Army  Medical  Corps,  edited 
by  Bruce. 

Valuable  papers  also  from  time  to  time  appear  in  the  Lancet,  Brit. 
Med.  Journ.,  Deutsche  med.  Wchnschr.,  Bcrl.  klin.  Wchnsclir.,  tiemaine 
med.,  Arch.  f.  Hyg.,  Arch.  f.  cxpcr.  Path.  u.  PharmaJcol.  Besides  these 
periodicals  the  student  may  have  to  consult  the  Reports  of  the  Med.  Off. 
of  the  Local  Government  Board,  which  contain  the  reports  of  the  medical 
officers,  also  the  Proc.  Roy.  Soc.  London,  the  Compt.  rend.  Acad.  d.  .ST. 
Paris,  the  Compt.  rend.  Soc.  de  biol.,  Paris,  and  the  Arb.  a.  d.  k.  Gsndht- 
samte.  (the  first  two  volumes  of  the  last  were  denominated  Mitthciluhgen}. 

For  general  reviews  on  Portozoal  and  Tropical  Diseases  generally,  see 
Manson,  "Tropical  Diseases,"  London,  1908;  and  Mense.  "  Handbuch 
der  Tropenkrankheiten,  Leipzig,  1906. 

CHAPTER  I. — GENERAL  MORPHOLOGY  AND  BIOLOGY. 

Consult  here  especially  Fliigge,  "Die  Mikroorganismen."  De  Bary, 
"Bacteria,"  translated  by  Garnsey  and  Bay  ley  Balfour,  Oxford,  1887. 
Zopf,  "Zur  Morphologic  der  Spalitpflanzen, "  Leipzig,  1882;  "  Beitr.  z. 
Physiologic  und  Morphologie  niederer  Organismen,"  5th  ed.,  Leipzig, 
1895.  Graham-Smith,  "  Parasitology,"  iii.  17.  Cohn,  Beitr.  z.  Biol. 
d.  Pflanz.,  Bresl.  (1876),  ii.  V.  Nageli,  "Die  niederen  Pilze,"  Munich, 
1877;  "  Untersuchungen  liber  niedere  Pilze/'  Munich,  1882.  Migula, 
"System  der  Bakterien,"  Jena,  1897.  Duclaux,  "Traite  de  micro- 
biologie,"  Paris,  1898-99.  For  general  morphological  relations,  see 
Marshall  Ward,  art.  "  Schizomycetes,"  Ency.  Brit.,  9th  ed.  xxi.  398; 
xxvi.  51.  Engler  and  Prantl,  "Die  natiirlichen  Pflanzenfamilien," 
Lieferung,  129.  "  Schizophyta  "  (by  W.  Migula).  STRUCTURE  OF  BAC- 
TERIAL CELL. — Biitschli,  "  Uber  den  Bau  der  Bakterien,"  Leipzig,  1890  ; 
"  Weitere  Ausfiihrungen  liber  den  Bau  der  Cyanophyceen  und  Bakterien," 
Leipzig,  1896.  Fischer,  op.  cit.  in  text.  Buchner,  Longard  and  Riedlin, 
Centralbl.  f.  Bakteriol.  u.  Parasitenk.  ii.  1.  Ernst,  Ztschr.  f.  Hyg. 
v.  428.  Babes,  ibid.  v.  173.  Neisser,  ibid.  iv.  165.  MOTILITY. — 
Klein,  Biitschli,  Fischer,  Cohn,  loc.  cit.  Loftier,  Centralbl.  f.  Bakteriol. 
u.  Parasitenk.  vi.  209  ;  vii.  625.  PIGMENTS.— Zopf,  loc.  cit.  ;  Galeotti, 
ref.  in  Centralbl.  f.  Bakteriol.  u.  Parasitenk.  xiv.  696.  Babes,  Ztschr. 


BIBLIOGRAPHY  651 

f.  ////'/.  xx.  3.  SPORULATION. — Prazmowski,  Biol.  Cent  mill.  viii.  301. 
A.  Koch.  Boto*.  Ztii.  (1888),  Nos.  18-22.  Buclmer,  Sitzunysb.  d.  math.- 
phys.  Cl.  d.  k.  buyer.  Akad.  d.  Wissmsch.  zu  Miinchen,  7th  Feb.  1880. 
II.  Kifh.  Mi'ffli.  '«.  d.  /,-.  (,'x)nth/mnnf>:  i.  65.  Dobell,  Quart.  Journ. 
Ml,-,-,  .sv.  (190i'\  liii.  CHKMICAL  STRUCTURE  OF  BACTERIA.  —  Nencki, 
/*'••/-.  'I.  d.-utsi'lt.  chfin.  Uwllxch.  (1884),  xvii.  2605.  Cramer,  Arch.  /. 
////.-/.  xvi.  1".4.  Buehner,  Bcrl.  klin.  //'.-A//..*-///-.  (1890),  673,  1084; 
rid,-  Klii^i:*',  "p.  fit.  CLASSIFICATION  OF  BACTERIA. — For  general  review, 

Marshall  Ward.  Ami.  of  Botany,  vi.  103;  Migula,  loc.  clt.  xti/n-n. 
!•'<••>!>  OF  HA-  rr.Kiv.  --Xiigeli,  Cohn,  op.  cit.  Pasteur,  "Etudes  sur  la 
bii'-iv,"  1S7«>.  Hiieppr,  Miftli.  a.  d.  k.  Gsndhtsamtc.  ii.  309.  RELATIONS 
TO  OXYCJKN.—  Pasteur,  t'ompt.  rend.  Acad.  d.  sc.  Hi.  344,  1142.  Kitasato 
and  \\Vyl.  Zlscli /-../'.  ////;/.  viii.  41,  404  ;  ix.  i»7.  TEMPERATURE.—  Vide 
Hiiirjr,..'  ,,y;.  c/V.  For  thermopliilic  bacteria,  Rabinowitsch,  Ztschr.  f. 
HIKJ.  \\.  l.")4.  Mai'fad\-en  and  Blaxall,  Journ.  Path,  and  Bacterial,  iii. 
-7.  ACTION  OF  BACTKEIAL  FERMENTS. — Salkowski,  Ztschr.  /.  Biol., 
N.  I-'.,  \  ii.  92.  Pasteur  and  Joubert,  Conijit.  rend.  Acad.  d.  sc.  Ixxxiii.  5. 
Shnidaii  Lea,  Jnnrn.  /'////x/W.  vi.  136.  Beijerinck,  Ccntralbl.  f.  Bak- 
t'  ,-lnf.  a.  I'aritsitenk.  Abth.  II.  i.  221.  E.  Fi.seber,  /ter.  ^.  deutsch.  chem. 

Vx.-//.  \\viii.  1430.  Liborius,  Xtx.hr.  f.  Hyy.  i.  115.  See  also 
Pasteur.  •' Royal  Society  Catalogue  of  Scientific  Papers."  Green,  "The 
Soluble  Ferinriits  and  Fermentation,"  Cambridge,  1899.  VARIABILITY. 
— Cohn,  Xii-^'li,  Kliigge,  op.  cit.  Winogradski,  "  Beitr.  •/..  Morph.  u. 
Pliysiol.  d.  Bakt./'  Leipzig,  1888.  Ray  Lankester,  Quart.  Joum.  M'n-r. 
,Vc.,  N.S.  (1873),  xiii.  408  ;  (1876),  xvi.  27,  278.  NITRIFYING  OROAMSMS. 
—Winogradski,  Ann.  de  VInst.  Pasteur,  iv.  213,  257,  760;  v.  92,  577. 
Ma/r.  ill,!.  \i.  11  ;  xii.  1,  263. 

CHAPTER  II.— METHODS  OF  CULTIVATION  OF  BACTEIMA. 

For  CKNKHAI.  PIMM  IIM.KS. — Pasteur,  Compt.  rend.  Acad.  d.  sc.  1. 
303  ;  Ii.  348,  675  ;  Ann.  de  chem.  Ixiii.  5.  Tyndall,  "Floating  Matter 
of  the  Air  in  Relation  to  Putrefaction  and  Infection,"  London,  1881. 
H.  C.  Bastian,  "The  Beginnings  of  Life,"  London,  1872.  METHODS  OF 
Si  I.I;II.ISATION.— R.  Koch,  Gatt'ky,  and  Li -tiler,  Mitth.  a.  d.  k.  Gsndhts- 
'•utte.  i.  322.  Koch  and  WolfFhiigel,  ibid.  i.  301.  CULTURE  MEDIA. 

S-i-  tr\t -Looks,  csj.oc-ially  Kanthack  and  Drysdale,  Eyre.  Pasteur, 
"  Btndea  sur  la  bi.'-re."  Paris,  1876.  R.  Koch,  Mitth.  a.  d.  k.  Gsndhts- 
<nn/,'.  i.  1.  Roux  et  Nocard,  Ann.  de  Vlnst.  Patteur,  i.  1.  Roux,  ibid. 
ii.  28.  Marmorek,  ibid.  ix.  593.  Kitasato  and  Weyl,  op.^  cit.  .supra.  P. 
and  Mrs.  Percy  Frankland,  "Micro-organisms  in  Water,"  London,  1894. 
Fuller,  Rep.  "Amer.  Pub.  Health  Ass.  xx.  381.  Theobald  Smith, 
Centralbl.  f.  Baktcriol.  u.  Parasitenk.  vii.  502 ;  xiv.  864.  Durham, 
Hi-it.  Med.  Journ.  (1898),  i.  1387.  "Report  of  American  Committee 
on  Bacteriological  Methods,"  Concord,  1898.  MacConkey,  Tkompson- 
}~,if,-s  ,i,id  Johnston  Lab.  Rep.  vol.  iii.  pt.  iii.  151  ;  vol.  iv.  pt.  i.  151  ; 
Journ.  Hyy.  v.  333.  Griinbaum  and  Hume,  Brit.  Mcd.  Journ. 
June  14,  1902.  Drigalski  and  Conradi,  Ztschr.  f.  Hyg.  xxxix.  283. 
Kudo,  <',  lttmm.  f.  Bakteriol.  u.  Parasitenk.  (Orig.),  xxxv.  109.  Con- 
radi. ;/,/,/.,  Beilage  zu.  Abth.  I.  Bd.  xlii.  (1908)  (Referate),  p.  *47. 
Fawcus,  Journ.  R.A.M.C.  xii.  147.  Sabouraud,  "  Les  Teignes, "  Paris, 
1910.  INDOL  REACTIONS.—  Bohme,  Centralbl.  /.  Bakteriol.  u.  Para- 
sitfiik.  Abth.  I.  (Orig.)  xl.  129.  Steensma,  ibid.  xii.  295.  Marshall, 
Journ.  Hyy.  vii.  581.  MacConkey,  ibid.  ix.  86. 


652  BIBLIOGRAPHY 

CHAPTER   III.— MICROSCOPIC  METHODS. 

Consult  text-books,  especially  Klein,  Kanthack  and  Drysdale,  Hueppe, 
Gimther,  Heim.  Also  Bolles  Lee,  "The  Microtomist's  Vademecum," 
London,  1905  (this  is  the  most  complete  treatise  on  the  subject). 
Rawitz,  op.  cit.  in  text.  Koch,  Mitth.  a.  d.  k.  Gsndhtsamte.  i.  1. 
Ehrlich,  Ztschr.  f.  Tclin.  Med.  i.  553;  ii.  710.  Gram,  Fortschr.  d. 
Med.  (1884),  ii.  No.  6.  Nicholle,  Ann.  de  I'lnst.  Pasteur,  ix.  666. 
Kiihne,  "  Praktische  Anleitung  zum  mikroscopischen  Nachweis  der 
Bakterien  im  tierischen  Gewebe,"  Leipzig,  1888.  Van  Ermengen,  ref. 
Centralbl.  f.  Bacterial,  u.  ParasitenTc.  xv.  969.  Richard  Muir,  Journ. 
Path,  and  Bacterial,  v.  374.  Mann,  "Physiological  Histology," 
Oxford,  1902.  For  Romanowsky  methods,  see  Jenner,  Lancet  (1899), 
i.  370.  Leishman,  Brit.  Med.  Journ.  (1901),  i.  635  ;  (1902),  ii.  757  ; 
Journ.  R.A.M.C.  (1904),  ii.  669.  Geimsa,  Deutsche  Med.  Wchnschr. 
(1905),  1026  ;  Ann.  de  I'lnst.  Pasteur,  xix.  346.  MacNeal,  Journ. 
Inf.  Diseases,  iii.  412.  Wright,  ,T.  H.,  Journ.  Med.  Research,  vii.  138. 
Wilson,  Journ.  Exp.  Med.  ix.  645. 

CHAPTER  IV.— METHODS  OF  EXAMINING  THE  PROPERTIES  OF  SERUM 
—  PREPARATION  OF  VACCINES  —  GENERAL  BACTERIOLOGICAL 
DIAGNOSIS — INOCULATION  OF  ANIMALS. 

AGGLUTINATION. — Delepine,  Brit.  Med.  Journ.  (1897),  ii.  529,  967. 
Widal  and  Sicard,  Ann.  de  I'lnst.  Pasteur,  xi.  353.  Wright,  Brit.  Med. 
Joum.  (1897),  i.  139  ;  (1898),  i.  355.  Park  and  Collins,  Journ.  Med. 
Research  (1904),  xii.  491.  Bainbridge,  Journ.  Path,  and  Baeteriol.  xiii. 
443.  Win  slow  and  Rogers,  "Biological  Studies  by  the  Pupils  of 
William  Thompson  Sedgwick,"  Boston,  1906. 

GENERAL  METHODS. — Wright,  A.  E.,  "Studies  on  Immunity,"  London, 
1909.  Muir,  Robert,  "Studies  on  Immunity,"  London,  1909.  Ehrlich, 
"  Gesammelte  Arbeiten  zur  Immunitiitsforschung,"  Berlin,  1904.  These 
works  contain  methods  applied  in  the  investigation  of  the  subjects  dealt 
with  in  this  chapter.  The  following  are  additional  references  relating  to 
special  points : — 

OPSOXIC  METHODS. — Klien,  H.,  John*  Hopkins  Hosp.  Bull.  (1907), 
xviii.  245.  Simon,  Journ.  Exp.  Med.  (1907),  487. 

WASSERMANN  REACTION. — Gengou,  Ann.  de  FInst.  Pasteur  (1902), 
xvi.  734.  Moreschi,  Berl.  Jclin.  Wchnschr.  (1905),  1181  ;  (1906),  100. 
Wassermann  and  Brack,  Deutsche  med.  Wchnschr.  (1906),  100.  Wasser- 
mann,  Neisser,  and  Bruck,  ibid.  (1906),  745.  McKenzie,  Journ.  Path,  and 
Baeteriol.  (1909),  xiii.  311.  Neisser,  Milnchen.  med.  Wchnschr.  (1909), 
No.  21,  1076. 

PREPARATION  OF  VACCINES. — Harrison,  Journ.  R.A.M.C.  (1905), 
iv.  313.  Leishman,  Harrison,  G rattan,  and  Archibald,  Hid.  (1908),  x. 
583  ;  (1908),  xi.  327. 

CHAPTER   V. — BACTERIA  OF  AIR,  SOIL,  WATER — 'ANTISEPTICS. 

AIR,  SOIL,  AND  WATER.— -Petri,  Ztschr.  f.  Hyg.  iii.  1  ;  vi.  233. 
Fliigge,  ibid.  xxv.  179.  Sticher,  ibid.  xxx.  163.  Weyl,  "Handbuch 
der  Hygiene,"  Jena,  1896,  et  seq.  Houston,  Rep.  Med.  Off.  Local  Gov. 
Bd.  xxvii.  (1897-98)  251  ;  xxviii.  (1898-99)  413,  439,  467  ;  xxix.  (1899- 
1900)  458,  489.  Sidney  Martin,  ibid.  xxvi.  (1896-97)  231  ;  xxvii. 


BIBLIOGRAPHY  653 

(1897-98)  308;  xxviii.  (1898-99)  382.  Horrocks,  "Bacteriological 
Examination  of  Water,"  London,  1901.  Percy  and  G.  C.  Frankland, 
" Micro-organ isma  in  Water,"  London,  1894.  Dibdin,  "  Purification  of 
Sewage  and  Water,"  London,  1897  ;  Ann.  Rep.  Bd.  Health,  Mass., 
Boston,  1890,  et  seq.  Savage,  "The  Bacteriol.  Exam,  of  Water  Supplies," 
London,  1906.  Lewis,  Rideal,  and  Walker,  Journ.  Roy.  San.  Inst. 
(1903),  xxiv.  424.  Prescott  and  Winslow,  "Elements  of  Water 
Bacteriology,"  New  York,  1908.  Houston,  "Annual  Reports  of  Metro- 
politan Water  Board,"  1907,  el  seq. ;  "Reports  on  Research  Work,  Metro- 
politan Water  Board,"  1907,  et  seq.  MacConkey,  Journ.  Hyg.  (1908), 
vol.  viii.  322  ;  (1909),  vol.  ix.  86.  Mair,  ibid.  (1908),  vol.  viii.  609. 
Lorrain  Smith,  "Third  Rep.  Roy.  Comm.  on  .Sewage  Disposal "  (1903),  ii. 
As  i  ISI.ITK  s.  —  R.  Koch,  Mitth.  a.  d.  k.  Gsndhtsamte.  i.  234.  Behring, 
/T/.v///-.  f.  Jlijij.  ix.  395.  Ritchie,  Trans.  Path.  Soc.  London,  1.  256. 
Rideal,  "Disinfection  and  Disinfectants,"  London,  1898.  -Chick  and 
Martin,  Journ.  Hi/if.  (1908),  vol.  viii.  654,  698.  Chick,  ibid.  vol. 
viii.  93. 

CHAPTER  VI. — RELATIONS  OF  BAOTKRIA  TO  DISEASE,  ETC. 

As  the  observations  on  which  this  chapter  is  based  are  scattered 
through  the  rest  of  the  book,  the  references  to  them  will  be  found  under 

the  ditl'crcnt  disra---. 

<  IIAPTER  VII.—  INFLAMMATORY  AND  SUPPURATIVE  CONDITIONS. 

Ogston,  11  fit.  Med.  Journ.  (1881),  i.  369.  Rosenbach,  "Mikro- 
organismen  bei  den  Wundinfectionskrankheiten  des  Menschen,"  Wies- 
baden, 1884.  Passet,  Fortschr.  d.  Med.  (1885),  Nos.  2  and  3.  W. 
Watson  Cheyne,  "Suppuration  and  Septic  Diseases,"  Edinburgh,  1889. 
Grawitz,  Virchow's  Archiv,  cxvi.  116  ;  Deutsche  med.  Wchnschr.  (1889), 
No.  23.  Steinhaus,  Ztschr.  f.  Hyg.  v.  518  (micrococcus  tetragenus) ; 
"Die  Aetiologie  der  acuten  Eiterung,"  Leipzig,  1889.  Christmas- 
Dirckinck-Holmfeld,  "  Recherches  expe>imentales  sur  la  suppuration," 
Paris,  1888.  Muir,  Journ.  Path,  and  Bacteriol.  vii.  161  ;  Trans.  Path. 
Soc.  London,  1902.  Garre,  Fortschr.  d.  Med.  (1885),  No.  6.  Marmorek, 
Ann.  dc,  VInst.  Pasteur,  ix.  593.  Petruschky,  Ztschr.  f.  Hyg.  xvii.  59; 
xviii.  413;  xxiii.  142  (with  Koch,  xxiii.  477).  Lubbert,  "  Biologische 
Spaltpilzuutersuchung,"  Wiirzburg,  1886.  Krause,  Fortschr.  d.  Med. 
(1884),  Nos.  7  and  8.  Ribbert,  ibid.  (1886),  No.  1.  Widal  and 
Bcsancon,  Ann.  de  VInst.  Pasteur,  ix.  104.  V.  Lingelsheim,  Ztschr. 
I.  Hyg.  x.  331  ;  xii.  308.  Behring,  Centralbl.f.  Bakteriol.  u.  Parasitenk. 
xii.  192.  Thoinot  et  Masselin,  Rev.  dc  med.  (1894),  449.  Orth  and 
Wyss.,k..\vits,-h,  Ccntralbl.  f.  d.  med.  Wisscnsch.  (1885),  577.  Netter, 
Arch.  'l<  jtlujxhJ.  norm,  rt path.  (1886),  106.  Weichselbaum,  Wien.  med. 
Wchnschr.  (1885),  No.  41  ;  (1888),  Nos.  28-32  ;  Central bl.  f.  Bakteriol.  u. 
Parasitenk,  ii.  209  ;  Beitr.  r.  path.  'Anat.  u.  c.  allg.  Path.  iv.  127. 
Becker,  Deutsche  med,  Wchnschr.  (1883),  No.  46.  Lannelongue  et 
Acliard,  Ana.  dc  VInst.  Pasteur,  v.  209.  Fehleisen,  "Die  Aetiologie 
des  Erysipel-v'  Berlin,  1883.  Welch,  Am.  Med.  Journ.  Sc.  (1891),  439. 
Lemoine,  Ann.  de  I  Inst.  Pasteur,  ix.  877.  Kurth,  Arb.  a.  d.  k. 
Gsndhtsamte.  vii.  389.  KnoiT,  Ztschr.  f.  Hyg.  xiii.  427.  Bnlloch, 
Lancet  (1896),  i.  982,  1216.  Bordet,  Ann.  de  VInst.  Pasteur,  xi.  177. 
Booker  (streptococcus  enteritis),  Johns  Hopkins  Hoxp.  Rep.  vi.  159. 


654  BIBLIOGRAPHY 

Hirsch,  Centralbl.  f.  Bakteriol.  u.  Parasitenk.  xxii.  369.  Libman,  ibid. 
xxii.  376.  Wright  and  Douglas,  Proc.  Roy.  Soc.  Loud.  Ixxiv.  147. 
Wright,  Clinical  Journal  (1906),  May  16. 

STREPTOCOCCI. — Hiss,  Journ.  Exper.  Med.  vi.  317.  Schottmiiller, 
Munchen.  med.  Wchnschr.  (1903),  849.  Gordon,  Reports  Med.  Officer 
Local  Gov.  Board  (1905),  388  ;  Lancet  (1905),  ii.  1400.  Andrewes  and 
Border,  Lancet  (1906),  ii.  Ruediger,  Journ.  Infect.  Diseases,  iii.  755. 
Besredka,  Butt,  de  I'Inst.  Pasteur,  iii.  Nieter,  Ztschr.  f.  Hyg.  (1907),  Ivi. 
307.  Mandelbaum,  ibid.  (1907),  Iviii.  26.  Levy,  Arch.  f.  path.  Anal. 
(1907),  ccxxxvii.  327.  Centralbl.  f.  Bakteriol.  u.  Parasitenk.  Abtheil  I. 
xliii.  793,  et  seq.  (hsemolytic  properties). 

CONJUNCTIVITIS. — Morax,  Ann.  d  I'Inst.  Pasteur  (1896),  x.  337.  Eyre, 
Journ.  Path,  and  Bacteriol.  vi.  1.  M tiller,  Wien.  med.  Wchnschr. 
1897  ;  Inglis  Pollock,  Trans.  Ophthalm.  Soc.  1905  ;  Axenfeld,  in  Lubarsch 
and  Ostertag,  "Ergebnisse  der  allgem.  Pathol.  u.  Path.  Anat.,"  1901  ; 
"  Die  Bakteriologie  in  der  Angenheilkunde,"  1907  (full  references). 

ACUTE  RHEUMATISM. — Tribouletand  Cayon,  Bull.  Soc.  med.  d.  h6p.  de 
Paris  (1898),  93.  Westphal,  Wassermann,  and  Malkoff,  Berl.  klin. 
Wchnschr.  (1899),  638.  Poyntoii  and  Paine,  Lancet  (1900),  ii.  861,  932 
(full  references).  Trans.  Path.  Soc.  Lond.  (1902),  liii.  221  ;  Lancet, 
December  1905.  Beaton  and  Walker,  Brit.  Med.  Journ.  (1903),  i.  237. 
Shaw,  Journ.  Path,  and  Bacteriol.  (1903),  ix.  158.  Beattie,  ibid.  ix.  272, 
xiv.  432  ;  Journ.  Med.  Research,  xiv.  399  ;  Journ.  Exper.  Med.  ix.  186. 
Cole,  Journ.  Infect.  Diseases,  i.  714.  Beattie,  Journ.  Path,  and  Bacteriol. 
xiv.  432. 


CHAPTER  VIII. — INFLAMMATORY  AND  SUPPURATIVE  CONDITIONS, 
CONTINUED  :  ACUTE  PNEUMONIAS,  EPIDEMIC  CEUEBRO  -  SPINAL 
MENINGITIS. 

Friedliinder,  Fortschr.  d.  Med.  i.  No.  22  ;  ii.  287  ;  Virclww's  Archiv, 
Ixxxvii.  319.  Fraenkel,  A.,  Ztschr.  f.  klin.  Med.  (1886),  401.  Salvioli 
and  Zaslein,  Centralbl.  f.  d.  med.  Wissensch.  (1883),  721.  Ziehl,  ibid. 
(1883),  433  ;  (1884),  97.  Klein,  ibid.  (1884),  529.  Jiirgensen,  Berl.  klin. 

Wchnschr.  (1844),  270.  Seibert,  ibid.  (1884),  272,  292.  Senger,  Arch, 
f.  exper.  Path.  u.  Pharmakol.  (1886),  389.  Weichselbaum,  Wien.  med. 

Wchnschr.  xxxvi.  1301,  1339,  1367;  Monatschr.  f.  Ohrenh.  (1888),  Nos. 
8  and  9  ;  Centralbl.  f.  Bakteriol.  u.  Parasitenk.  v.  33.  Gamaleia,  Ann. 
de  I'Inst.  Pasteur,  ii.  440.  Guarnieri,  Atti  d.  r.  Accad.  med.  di  Roma 
(1888),  ser.  ii.  iv.  Kruse  and  Pansini,  Ztschr.  f.  Hyg.  xi.  279.  E. 
Fraenkel  and  Reiche,  Ztschr.  f.  klin.  Med.  xxv.  230.  Sanarelli,  Centralbl. 
f.  Bakteriol.  u.  Parasitenk.  x.  817.  Launelongue,  Gaz.  d.  hop.  (1891), 
379.  Netter,  Bull,  et  mem.  Soc.  med.  d.  h6p.  de  Paris  (1889)  ;  Compt. 
rend.  Acad.  d.  sc.  (1890) ;  Compt.  rend.  Soc.  de  biol.  Ixxxvii.  34. 
G.  and  F.  Klemperer,  Berl.  klin.  Wchnschr.  (1891),  869,  893.  Foa  and 
Bordoni-Uffreduzzi,  Deutsche  med.  Wchnschr.  (1886),  No.  33.  Emmerich, 
Milnchen.  med.  Wchnschr.  (1891),  No.  32.  Issaeff,  Ann.  de  I'Inst. 
Pasteur,  vii.  260.  Grimbert,  Ann.  de  I'Inst.  Pasteur,  xi.  840.  Wash- 
bourn,  Brit.  Med.  Journ.  (1897),  i.  510 ;  (1897),  ii.  1849.  Eyre  and 
Washbourn,  Journ.  Path,  and  Bacteriol.  iv.  394  ;  v.  13.  See  also 
Brit.  Med.  Journ.  (1901),  ii.  760.  Neufeld  and  Rimpau,  Ztschr.  f.  Hyg. 
Ii.  283.  Rosenow,  Journ.:  Am.  Med.  Ass.  (1908),  Ii.  No.  19.  Neufeld  and 
Handel,  Ztschr.  f.  Immunitdtsforschung  (1909),  iii.  159.  Tschistowitch  and 
Jurewitch,  Compt.  rend.  Soc,  de  biol.  (1908),  Ixiv.  pp.  1044,  1095. 


BIBLIOGRAPHY  655 

Commission    to     Invi-stigate   Acute   Resp.    Dis.   (Hiss   and    others),    see 
Jour*.  A'./-//.  M'd.  (1905),  vii.  pp.  403-632. 

MK\IM;ITIS.— Weichselbauiu,  Fortschr.  d.  Med.  (1887),  v.  573,  620. 
JHCIOT.  Xtxi-hr.f.  Ifii'.i.  xix.  351  ;  xliv.  225.  Councilman,  Mallory,  and 
Wright.  "  Epidemic  Cerebro-spinal  Meningitis,"  Rep.  Bd.  Health,  Mass., 
.11,  1898  (full  references).  Gwyn,  Johns  Hopkins  Hosp.  Bull.  (1899), 
109.  v.  Lingt'lslu'ini,  Klin.  Jahrb.  xv.  373.  Kolle  and  Wassermann, 
ibul.  p.  507.  Kutscher,  Deutsche  med.  Wchnschr.  (1906),  1071.  Betteu- 
court  and  Franca,  Ztschr.  f.  Hyg.  xlvi.  463.  Durham,  Journ.  Infect.  Dis. 
XniH>1.  No.  2,  p.  10.  Goodwin  and  von  Sholly,  ibid.  p.  21.  Arkwriglit, 
Jnurn.  nf  J/fft/.  vii.  145  ;  ix.  104.  Flexner,  Journ.  Exper.  Med.  ix.  105. 
Klt-xner  and  jobling,  ibid.  x.  141,  690.  Vansteenberghe  et  Grysez,  Ann. 
</<  ////.>/.  /v/.sV,  ///•.  xx.  69.  Gordon,  Report  to  Local  Govt.  Board  on  the 
Mi(  rococcus  of  Cerebro-spinal  Meningitis,"  London,  H.M.  Stationery  Office, 
1907.  Albrecht  and  Ghou,  Wien.  Tclin.  Wchnschr.  (1901),  xiv.  984  ; 
/,'  /-.  Newr.  and  I'^ichlat.  (1907),  v.  593,  686.  Stuart  M 'Donald,  Journ. 
/'»th.  //,///  Bncteriol.  (1908),  xii.  442.  Shennan  and  W.  T.  Ritchie,  ibid. 
xii.  4f>(5.  M'Kenxie  and  Martin,  ibid.  xii.  539.  J.  Ritchie,  ibid. 
(1910),  xiv.  615.  Elser  and  Huntoon,  Journ.  Med.  Research  (1909),  xx. 
371.  Discussion  in  Brit.  Med.  Journ.  (1908),  ii.  1334. 

CHAPTER  IX. — GONORRHCEA,  SOFT  Son i.. 

<;<>N«II;KH<KA. — Neisser,  Centralbl.  f.  d.  med.  Wissensch.  (1879),  497  ; 
lt,-nt*'lt,<  »ned.  Wchnachr.  (1882),  279  ;  (1894),  335.  Bumm,  "Der  Mikro- 
or^anismus  der  gonorrhoischen  Schleimhauterkrankungen,"  Wiesbaden, 
1885,  2nd  ed.  1887  ;  Miinchcn.  med.  Wchnschr.  (1886),  No.  27  ;  (1891), 
Nos.  50  and  51  ;  Centralbl.  f.  Gyndk.  (1891),  No.  22  ;  Wien.  med.  Presec 
(1891),  No.  24.  Bockhart,  Monatsh.  f.  prakt.  Dcrmat.  (1886),  v.  No.  4  ; 
(1887),  vi.  No.  19.  Steinschneider,  Berl.  klin.  Wchnschr.  (1890),  No. 
•Jl  ;  H893),  No.  29  ;  Verhandl.  d.  deutsche  dermat.  Gescllsch.  I.  Congress, 
Wirn  (1889),  159.  Wertlu-im,  U'icn.  klin.  Wchnschr.  (1890),  25  ; 
It. -nfv-fte  med.  ll'<-hnschr.  (1891),  No.  50  ;  Arch.  f.  Gyniik.  xii.  Heft  1  ; 
CetitraW.  f.  (.'••fl,«k.  (1891),  No.  24;  (1892),  No.  20;  Wien.  klin. 
FTdbudtr.  (1894),  441.  Gerhardt,  Charity-Ann.  (1889),  241.  Leyden, 
Zf.«rlu:  f.  klin.  ^f«/.  xxi.  607;  Deutsche,  med.  Wchnschr.  (1893),  909. 
Bordoni-Uflreduzzi,  ibid.  (1894),  484.  Councilman,  Am.  Journ.  Med. 
A'--.  <-vi.  277.  Finger,  Ghou,  and  Schlagenhaufer,  Arch.  f.  Dermat.  ». 
,S'///'//.  xxviii.  3,  276.  Lang,  ibid.  (1892),  1007;  Wien.  med.  Wchnschr. 
M>!'i;,  No.  7;  "Der  Venerische  Katarrh,  dessen  Pathologic  und 
Therapie,"  Wit-sluulcn,  1893.  Klein,  Mmmtschr.f.  Gcburtsh.  u.  Gynaek. 
(1895),  33.  Michaelis,  Ztschr.  f.  klin.  Med.  xxix.  556.  Heimaii,  New 
York  Med.  Rec.  (1895),  769  ;  (1896),  Dec.  19.  Foulerton,  Trans.  Brit. 
Inst.  Prer,  ,i .  M,,l.  \.  40.  De  Christmas,  Ann.  de  Vlnst.  Pasteur,  xi. 
609.  Nicolaysen,  Centralbl.  f.  Bakteriol.  u.  Parasitenk.  xxii.  305. 
K.-ndu,  /-''/•/.'/.•/;//.  Jl'r/inschr.  (1898).  431.  Wassermann,  Ztschr.  f. 
////v.  xx  vii.  2!»s  :  .!/;/,/«•//»•,/.  ,,inl.  )lrchnschr.  (1901),  No.  8.  Leiihartz, 
/;.'/•/.  /•////.  Wchiwhr.  (1897),  1138.  Thayer  and  Lazear,  Journ.  Exper. 
.]/<->/.  iv.  81.  Kiinig.  Berl.  klin.  Wchnschr.  (1900),  No.  47..  De  Christmas, 
Ann.  '/.  /'Inst.  Pasteur  (1900),  xiv.  331.  Raskai,  Deutsche  med.  WchMclir. 
(1901),  No.  1.  Jundell,  Centralbl.  f.  Bakteriol.  u.  Parasitenk.  xxix. 
•J-J4.  ("lo ml. ini,  ibiil.  xxiv.  955.  Hrcssel,  Miinchen.  med.  Wchnschr. 
(1903\  No.  13.  Moller,  Arch.  f.  Dermat:  u.  Syph.  (1904),  Ixxi.  269. 
Wyiin.  Lnn'-.t  (1905),  i.  352.  Prochaska,  Arch.  f.  klin.  Med.  Ixxxiii. 


656  BIBLIOGRAPHY 

Heft  1-2.  Strong,  Journ.  Am.  Med.  Ass.  May  1904.  Gurd,  Journ.  Med. 
Research  (1908),  xviii.  291.  Th.  Vanned,  Centralbl.  f.  Bakteriol.  u. 
Parasitenk.  Abth.  I.  (Orig.)  (1907),  xliv.  10, 110.  Hamilton,  Journ.  Infect. 
Diseases  (1908),  v.  133.  Brons,  Klin.  Monatsbl.  f.  Augenheilk.  (1907), 
xlv.  1.  Torrey,  Journ.  Med.  Research  (1908),  xix.  471.  Martin,  Journ. 
Path,  and  Bacterial.  (1910),  xv.  76. 

SOFT  SORE. — Ducrey,  Monatsh.  f.  praTct.  Dermat.  ix.  221.  Krefting, 
Arch.  f.  Dermat.  u.  Syph.  (1892),  263.  Jullien,  Journ.  d.  mal.  cutan.  et 
syph.  (1892),  330.  Unna,  Monatsh.  f.  prakt.  Dermat.  (1892),  475  ;  (1895), 
61.  Quinquand,  Semaine  mid.  (1892),  278.  Petersen,  Centralbl.  f. 
Bakteriol.  u.  Parasitenk.  xiii.  743  ;  Arch.  f.  Dermat.  n.  Syph.  (1894), 
419.  Audrey,  Monatsh.  f.  prakt.  Dermat.  (1895),  267.  Colombini, 
Centralbl.  f.  Bakteriol.  u.  Parasitenk.  xxv.  254.  Nicolle,  Presse  medicale 
(1900),  304.  Bezan9on,  Griifon,  and  Le  Sourd,  Ann.  de  dermat.  et  de 
syphilolog.  (1901),  tome  ii.  1.  Lenglet,  ibid.  (1901),  tome  ii.  209.  Simon, 
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(1903),  Bd.  43,  p.  327.  Davis,  Journ.  of  Med.  Research  (1903),  ix.  401. 

CHAPTER  X.— TUBERCULOSIS. 

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ausd.  kaiserl.  Gsndhtsmte.,  Berlin,  1904-1905.  Weber  and  Tante,  ibid. 
1905.  Salmon  and  Smith,  "Tuberculosis,"  U.S.  Department  of  Agri- 
culture, Washington,  1904.  Wolbach  and  Ernst,  Journ.  Med.  Research, 
x.  313.  "  Interim  Reports  of  the  Royal  Commission  on  Tuberculosis," 
London,  1904,  1907.  Wright  and  Douglas,  Proc.Roy.  Soc.  Lond.,]xxiv. 
159.  Wright,  Clinical  Journal,  Nov.  9,  1904;  ibid.,  May  15,  1906; 
Med.  Chir.  Tran.  (1905)  Ixxxix.  Wright  and  Reid,  Proc.  Hoy.  Soc. 
Loud.,  Ixxvii.  194,  211.  v.  Pirquet,  Berlin,  klin.  Wchnschr.  (1907).  Vide 
also  article  on  "  Kutane  u.  konjunktivale  Tuberkulinreaktion,"  in  Kraus 
.ind  Levaditi's  Techniku.  Methodik  der  Immunitdtsforschung,  Bd.  I.  1035. 
Wolf!'-  Eisner,  Berlin,  klin.  Wchnschr.  (1907).  Calmette,  Compt.  rend. 
Acad.  d.  Sciences  (1907),  1324.  Calmette,  Breton,  Painblon  et  Petit, 
J'ri'sse  med.  (1907),  xv.  443.  Petit,  "  Le  diagnostic  de  la  tuberculose  par 
I'ophthalmo-reaction"  (full  references),  Paris,  1908.  Much,  Beitr.  Klin.  d. 
Tuberc.  (1907),  viii.  85,  99,  357-  Wirths,  Miinchen.  med.  Wchnschr. 
(1908),  Iv.  1687.  Trauholz,  New  York  Med.  Rcc.  (1908),  60.  Herman,  Ann. 
de  I'Inst.  Pasteur,  xxii.  92.  Frugoni,  Centralbl.  f.  Bakteriol.  u.  Parasitenk. 
(Orig.)  (1910),  liii.  553.  Twort,  Proc.  Roy.  Soc.  Lond.,  B.  Ixxxi.,  March 
1909. 

acid-fast  bacilli.—  Moeller,  Deutsche  med.  Wchnschr.  (1898),  376. 
.  Bakteriol.  u.  Parasitenk.  xxv.  369;  ibid.  xxx.  513.  Petri, 
Arl>.  a.  d.  k.  Gsndhtsamlc.  (1898),  1.  Rabinowitch,  Deutsche  med. 
H'cluwhr.  (1897),  No.  26;  (1900),  No.  16;  Ztschr.  f.  Hyg.  xxvi.  90. 
Koi'ii,  Arch.  f.  Hyg.  xxxvi.  57;  Centralbl.  f.  Bakteriol.  u.  Parasitenk. 
\\vii.  481.  Schulze,  Ztschr.  f.  Hyg.  xxxi.  153.  M.  Toblcr,  ibid,  xxxvi. 
120.  Lubarsch,  ibid.  xxxi.  'l87.  Holscher,  Centralbl.  f.  Bakteriol.  u. 
1',1,-nsif,  'nJ:.  xxix.  425.  Potet,  "  fitude  sur  les  bacilles  dites  'acido- 
philes,'"  Paris,  1902.  Abbott  and  Gildersleeve,  Univ.  of  Pennsylvania 
M.il.  /;////<•////.,  June  1902  Johne  and  Frothingham,  Deutsche  Ztschr.  f. 
T/i  termed.  (1895),  438.  McFadyean,  Journ.  Compar.  Path.  xx.  (1907),  48. 
Tliilibert,  "Les  pseudo-bacilles  acido-resistants,"  Paris,  1908. 

CHAPTER  XI.—  LEPROSY. 

Hansen,  Norsk.  Mag.  f.  L«gevidensk.,  1874;  Virtliows  Archir,  Ixxix. 
:\-2  ;  xc.  542  ;  ciii.  388  ;  Virchow's  Feslschr.  (1892),  iii.  See  papers  by 
Ni-isser  and  Cornil  and  Suchard  in  "  Microparasites  in  Disease"  (New 
Soc.,  1886).  Hansen  and  Looft,  "  Leprosy,"  Bristol,  1895. 


42 


658  BIBLIOGRAPHY 

Doutrelepont  and  "VVolters,  Arch.  f.  Dermat.  u.  Syph.  (1892),  55. 
Thoma,  Sitzungsb.  d.  Dorpater  Naturforsch.,  1889.  Unna,  Dermat. 
Stud.  Hamburg  (1887),  iv.  Bordoni-Uff'reduzzi,  Ztschr.  f.  Hyg.  iii. 
178 ;  Berl.  klin.  Wchnschr.  (1885),  No.  11.  Avning  and  Nonne, 
Virchow's  Archiv,  cxxxiv.  319.  Gairdner,  Brit.  Med.  Journ.  (1887), 
i.  1296.  Hutchinson,  Arch.  Surg.  (1889),  i.  v.  Torok,  "Summary  of 
Discussion  on  Leprosy  at  the  first  Internat.  Congr.  for  Dermatol.  and 
Spyh."  v.  Moiiatsh.  f.  prakt.  Dermat.  ix.  238.  Profeta,  Gior.  intcrnaz. 
d.  sc.  med.,  1889.  See  Journal  of  the  Leprosy  Investigation  Committee, 
1890-91.  Philip pson,  Virchow's  Archiv,  cxxxii.  529.  Daniclssen, 
Monatsh.  f.  prakt.  Dermat.  (1891),  85,  142.  Wesener,  CcntralbL  f. 
BakterioL  u.  Parasitenk.  ii.  450  ;  Miinchen.  mcd.  Wchnschr.  (1887), 
No.  18.  Uhleuhuth  and  Westphal,  Centralbl.  f.  BakterioL  u.  Parasitenk. 
xxix.  233.  Babes  in  "  Erganzungsband "  of  Kolle  and  Wassermann's 
Handbuch  dcr  Pathogenen  Mikro-organismen.  Dean,  Journ.  of  Hyg.  v. 
99.  Wherry,  Journ.  Infect.  Diseases  (1908),  v.  507.  Kitasatoj  Ztschr.  f. 
Hyg.  (1909),  Ixiii.  507.  March oux  and  Bourret,  Ann.  de  f'lnst.  Pasteur 
(1909),  xxiii.  513.  Clegg,  Philippine  Jour.  Sc.,  Series  B.  iv.  (1909). 
Slatineano  and  Danielopolu,  Compt.  rend.  Soc.  biol.  (1908),  Ixv.  347  ; 
(1909),  Ixvi.  332. 

CHAPTER  XII.— GLANDERS  AND  RHINOSCLEROMA. 

Loffler  and  Schultz,  Deutsche  med.  Wchnschr.  (1882),  No.  52.  Loftier, 
Mitth.  a.  d.  k.  Gsndhtsamte.  i.  134.  Weichselbaum,  Wien.  med. 
Wchnschr.  (1885),  Nos.  21-24.  Preusse,  Berl.  thierarztl.  Wchnschr. 
(1889),  Nos.  3,  5,  11  ;  ibid.  (1894),  Nos.  39,  51.  Gamaleia,  Ann.  de 
Vlnst.  Pasteur,  iv.  103.  A.  Babes,  Arch,  de  med.  exper.  et  d'anat.  pat/t. 
(1892),  450.  Straus,  Compt.  rend.  Acad.  d.  sc.  cviii.  530.  M'Fadyean 
and  Woodhead,  Hep.  National  Vet.  Assoc.,  1888.  Baumgarten,  Centralbl. 
f.  BakterioL  u.  Parasitenk.  iii.  379.  Silviera,  Semainc  mtd.  (1891), 
No.  31.  Bonome,  Deutsche  med.  Wchnschr.  (1894),  703,  725,  744. 
Kalning,  Arch.  f.  Veterindrwissensch.  (St.  Petersburg),  i.  Apr.  May. 
Foth,  Centralbl.  f.  BakterioL  u.  Parasitenk.  xvi.  508,  550.  M'Fadyean, 
Journ.  Comp.  Path,  and  Therap.,  1892,  1893,  1894.  Leclainche  and 
Montane,  Ann.  de  Vlnst.  Pasteur,  vii.  481.  Leo,  Ztschr.  f.  Hyg.  vii. 
505.  Marx,  Centralbl.  f.  BakterioL  u.  Parasitenk.  xxv.  275.  Mayer, 
ibid,  xviii.  673.  Bonome,  Centralbl.  f.  BakterioL  u.  Parasitenk.  (Reler.), 
xxxviii.  97.  Anderson,  Chalmers,  and  Buchanan,  Glasgow  Med.  Journ., 
Oct.  1905.  Nicolle,  Ann.  de  Vlnst.  Pasteur,  xx.  623,  698,  801  ;  ibid.  (1907), 
xxi.  281.  Schnurer,  Centralbl.  f.  BakterioL  u.  Parasitenk.  (Refer.), 
(1909),  xlii.  :  Supplem.  167  ;  Ztschr.  f.  Infektionskrank.  d.  Hausthiere. 
(1908),  iv.  216.  Collins  (agglutination),  Journ.  Infect.  Diseases  (1908),  v. 
401.  Miiller,  Ztschr.  f.  Immnnitdtsf.  (Orig.)  (1909),  iii.  401.  Valenti, 
ibid.  (1909),  98. 

RHINOSCLEROMA.— Frisch,  Wien.  med.  Wchnschr.  (1882),  No.  32. 
Cornil  and  Alveraz,  Arch,  de  physiol.  norm,  et  path.  (1895),  3rd  series, 
vi.  11.  Paltauf  and  Eiselsberg,  Fortschr.  d.  med.  (1886),  Nos.  19,  20. 
Wolkowitsch,  Centralbl.  f.  d.  med.  Wisscnsch.  (1886).  Dittrich,  Ztschr. 
f.  Heilk.  viii.  251.  Babes,  Centralbl.  f.  BakterioL  u.  Parasitenk.  ii. 
617.  Pawlowski,  ibid.  ix.  742;  "  Sur  1'etiologie  et  la  pathologic  du 
rhinosclerome,"  Berlin,  1891.  Paltauf,  Wien.  med.  Wchnschr.  (1891), 
Nos.  52,  53;  (1892),  Nos.  1,  2.  Wilde,  Semaine  med.  (1896),  336. 
Klemperer  and  Scheier,  Ztschr.  f.  klin.  Med.  xlv.  Heft  1-2.  Lanzi, 


BIBLIOGRAPHY  r>59 


Cfitfralbl.f.  H.il-trri,,/.  a.  /Wr/N/7,  ,//•.  (KdV-r.),  \x.\iv.  «!27.     SehaMowski, 
ibid,  \\xviii.  714.     Perkins,  Jo  urn.  Infect.  Diaauct  (1907),  iv.    51. 

CHAPTER  XIII.—  ACTINUMV.  osis,   KT«  . 

llollinger,  <  '<  -uti-nllil.  f.  d.  tncd.  IV-isscHsck.,  1877.  J.  Israel,  F/n7/«//-'.s- 
Ari'hir.  Ixxiiv.  15;  Ixxviii.  421.  Ponfick,  Brcxlnn.  aertol.  Z(*>-/,,:,  : 
"Die  Aktinomykose  des  Menschen,"  1882.  0.  Israel,  Vircltmr's  Arc/iir, 
xcvi.  17"'.  Chiari,  Pray.  med.  Wchnschr.,  1884.  Langhans,  t'or.-Bl.  /'. 
sr/.  //•/»/;.  .lcr-Jc  (1888),  xviii.  Liining  and  Hanau,  ibid.  (1889),  xix. 
Shattook,  Trans.  Path.  Soc.  London,,  1885.  Aclaud,  ibid.  1886.  Delepine. 
//>/</.  ISK'.i.  Harley.  M.d.-Chir.  Trans.,  London,  1886.  Crookshank., 
ibiil.  1889;  "Manual  of  Bacteriology,"  London,  1896.  Ransome,  J/"/.- 
Chir.  Trans.,  London,  1891.  M'Fadyean,  Journ.  Comp.  Path,  mxl 
Th-  r>'i>.,  1889.  Bostroni.  Beitr.  t.  path.  Anat.  •//.  c.  a////.  Pa//t.,  1890. 
Woltt'  and  Israel,  Virclioid's  Archiv,  cxxvi.  11.  Illich.  ''Beitrage  xur 
Klinik  der  Aktinomykose,"  Wien,  1892.  Grainger  Stewart  and  Muir, 
K'f.i.  J/ns/t.  ];<-p.,  1893.  Leith,  ibid.  1894.  Gasperini,  Centralbl.  f. 
Baktfi-lot.  >'.  I'n  I'tixitc  nk.  xv.  684.  Hummel,  Beitr.  z.  klin.  Chir.  xiii. 
No.  3.  Pawlowsky  and  Maksutoff,  Ann.  dc  I'Inst.  Pasteur,  vii.  544. 
Nenkirch,  Uebcr  Mrahlcnjrilze,  Strassburg,  1902.  Doepke,  M  a  m-l  •••>!. 
///"/.  11',-hnsrhr.i  1902.  Sillurschmidt,  Ztschr.  f.  Hyy.  xxxvii.  345. 
.1.  Homer  Wright,  Publications  of  the  Massachusetts  General  Ho.y/fn/, 
liuston,  May  1905.  Neuhaiiser,  Deutsche  med.  Wchnschr.  (1907),  1457. 
\Vi)o!ilridge,  Jimrn.  Compar.  Path,  and  Therap.  (1907),  xx.  Fritzsche, 
A  >•<•/,  iv.f.  Hyy.  (1908),  Ixv.  181. 

Allied  Mrcptothrices;—'8ocsird,  Ann.  de  I'Inst.  Pasteur  (1888),  ii.  293. 
Kppinger,  Beitr.  z.  path.  Anat.  u.  c.  allg.  Path.  ix.  287  ;  in  Lubarsch  and 
Ostortag,  "  Krgebnisse  der  allgem.  Path."  iii.  328.  Buchholz,  Ztschr.  f. 
////.'/.  xxiv.  470.  Berestnew,  ibid.  xxix.  94.  Cozzolino,  ibid,  xxxiii.  36. 
Flexner,  Journ.  E.'-^r.  Med.  iii.  435.  Dean,  Trans.  Path.  Soc.  London 
(1900),  26.  Birt  and  Leishman,  Journ.  of  Hyg.  ii.  120.  Mertens, 
'  '.  ,/t,  -it/hi,  f.  Bakteriol.  u.  Parasitenk.  xxix.  694.  Foulerton,  Trans. 
I'ntli.  S»c.  'London  (1902),  56.  M'Donald,  Trans.  Med.  -Chir.  Soc.  Edin. 
xxiii.  131.  N  orris  and  Larkins,  Journ.  Exper.  Med.  v.  155.  Butter- 
field,  Journ.  Infect.  Diseases,  vi.  421.  Litten  and  Levy,  Deutsche  med. 
//W/,/.sr///\  (1906),  1772. 

MADTKA  DISKASK.  —  Carter  "On  Mycetoma  or  the  Fungus  Disease  of 
India,"  London.  Ba.ssini,  ref.  in  Ccntralbl.f.  Bakteriol.  u.  Parasitenk. 
iv.  652.  Lewis  and  Cunningham,  Eleventh  Ann.  Rep.  San.  Com.  India. 
K.'.bner,  Fortschr.  d.  Med.  (1886),  No.  17.  Kanthack,  Journ.  Path,  and 
Ji'H-trriol.  \.  140.  Boyee  and  Surveyor,  Proc.  Roy.  Soe.  London,  1893. 
Vandyke  Carter,  Trans.  Path.  Soc.  London,  1886.  Vincent,  Ann.  </<• 
/'/nut.  Pasteur,  viii.  129.  J.  H.  Wright,  Journ.  Exper.  Med.  iii.  421. 
Oppenheim,  Arch.  f.  Dermat.  u.  Syph.  Jxxi.  209.  Bruin  pt,  "  Les 
.My  '-tomes,"  Paris,  1906. 

CHAPTER  XIV.—  ANTHRAX. 

Bellinger  in  Ziemssen's  "  Cycloptedia  of  Medicine."  Greenfield, 
"Malignant  Pustule/'  in  Qnaut'a  "Dictionary  of  Medicine,"  London, 
1894.  Pollender,  I'rtljschr.  f.  [jcrlchtl.  Med.  viii.  Davaine,  Compt. 
r,  ,i,l.  Acad.  d.  sc.  Ivii.  220,  351,  386  ;  lix.  393.  Koch,  Cohn's  Beitr.  z. 
l.  d.  Pftanz.  (1876),  ii.  Heft  2  ;  Mitth.  a.  d.  k.  Gsndhtsamtf..  i.  49. 


660  BIBLIOGRAPHY 

Pasteur,  Compt.  rend.  Acad.  d.  sc.  xci.  86,  455,  531,  697  ;  xcii.  209. 
Buchner,  Virchow's  Archiv,  xci.  Chamberland,  Ann.  de  I'lnst.  Pasteur, 
viii.  161.  Chauveau,  Com.pt.  rend.  Acad.  d.  sc.  xci.  33,  648,  880  ;  xcvi. 
553.  Czaplewski,  Beitr.  z.  path.  Anat.  u.  z.  allg.  Path.  vii.  47. 
Gamaleia,  Ann.  de  I'lnst.  Pasteur,  ii.  517.  Marshall  Ward,  Proc.  Roy. 
Soc.  London,  Feb.  1893.  Petruschky,  Beitr.  z.  path.  Anat.  u.  z.  allg. 
Path.  iii.  357.  Weyl,  Ztschr.  f.  Hyg.  xi.  381.  Behring,  ibid.  vi.  117  ; 
vii.  171.  Osborne,  Arch.  f.  Hyg.  xi.  51.  Roux,  Ann.  de  I'lnst.  Pasteur, 
iv.  25.  Hankin,  Brit.  Med. '  Journ.  (1889),  ii.  810;  (1890),  ii.  65. 
Hankin  and  Wesbrook,  Ann.  de  I'lnst.  Pasteur,  vi.  633.  Sidney  Martin, 
Rep.  Med.  Off.  Local  Govt.  Board  (1890-91),  255.  Marmier,  Ann.  de  I'lnst. 
Pasteur,  ix.  533.  Rd.  Muir,  Journ.  Path,  and  Bacterial,  v.  374.  Sclavo, 
Rivista  d'Igiene  e  Sannita  pubblica,  vii.  Nos.  18,  19 ;  Sulla  stato 
presente  delict  Sieroiherapia  anticarbonchiosa.  Turin,  Pozzo,  1903  (see 
Legge,  Lancet  (1905),  i.  689,  765,  841).  Sobernheim  in  Kolle  and  Wasser- 
niann's  Handbuch,  iv.  793.  Cler,  Centralbl.  f.  Bakteriol.  u.  Para- 
sitenk.  (Orig.)  xl.  241.  Bail,  ibid,  xxxiii.  343,  610.  Saufelice,  ibid. 
xxxiii.  61.  Roger  and  Gamier,  Compt.  rend.  Soc.  de  liol.  Iviii.  863. 
Teacher,  Lancet  (1906),  i.  1306.  M'Fadyean,  Journ.  Comp.  Path.  (1903), 
xiv.  35,  360.  Cave,  ibid.  (1908),  320.  Heim,  Archiv.  f.  Hyg.  xl.  55. 
Sobernheim  in  Kraus  and  Levaditi's  "  Handbuch  der  Technik  und 
Methodik  der  Immunitiitsforschung,"  Jena,  1908,  ii. 

CHAPTER  XV.— TYPHOID  FEVER,  ETC. 

BACILLUS  COLT. — Escherich,  Centralbl.  f.  Bakteriol.  u.  Parasitenk. 
(1887),  i.  705  ;  ibid.  (1888),  iii.  675,  801  ;  Deutsche med.  Wchnschr.(\888), 
No.  24.  Gordon,  Journ.  Path,  and  Bacteriol.  iv.  438.  MacConkey, 
Journ.  of  Hyg.  (1905),  v.  333  ;  (1906),  vi.  385  ;  (1909),  ix.  86.  Wilson, 
ibid.  (1908),  viii.  543.  Prescott  and  Winslow,  "  Elements  of  Water 
Bacteriology,"  New  York,  1908.  Voges  and  Proskauer,  Ztschr.  f.  Hyg. 
(1898),  xxviii.  20. 

EARLY  WORK  ON  B.  TYPHOSUS. — Eberth,  Virchow's  Archiv,  Ixxxi.  58  ; 
Ixxxiii.  486.  Koch,  Mitth.  a.  d.  k.  Gsndhtsamte.  i.  46.  Galfky,  ibid. 
ii.  80.  Klebs,  Arch.  f.  exper.  Path.  u.  Pharmakol.  xii.  231  ;  xiii.  381. 
Escherich,  Fortschr.  d.  Med.  (1885),  Nos.  16,  17.  Emmerich,  Arch.  f. 
Hyg.  iii.  291.  Rodetand  Roux,  Arch,  de  'intd.  exper.  et  d'anat.  path.  iv. 
317.  Weisser,  Ztschr.  f.  Hyg.  i.  315.  Klein,  "Micro-organisms  and 
Disease,"  London,  1896  ;  Rep.  Med.  Of.  Local  ffovt.  Board  (1892-93),  345  ; 
(1893-94),  457  ;  (1894-95),  399,  407,  411.  Babes.  Ztschr.  f.  Hyg.  ix. 
323.  Vincent,  Compt.  rend.  Soc.  de  biol.  se"r.  ix.  ii.  62.  Birch -Hirseh- 
feld,  Arch.  f.  Hyg.  vii.  341.  Buchner,  Centralbl.  f.  Bakteriol.  u.  Para- 
sitenk. iv.  353.  Pfuhl,  ibid.  iv.  769.  Petruschky,  ibid.  vi.  660.  Hunter, 
Lancet  (1901),  i.  613.  Kitasato,  Ztschr.  f.  Hyg.  vii.  515.  Chantemesse 
and  Widal,  Bull.  med.  (1891),  No.  82  ;  Ann.  de  TImt.  Pasteur,  vi.  755  ; 
vii.  141.  Pere,  Ann.  de  I'lnst.  Pasteur,  vi.  512.  Neisser,  Ztschr.  f.  klin. 
Med.  xxiii.  93.  Nicholle,  Ann.  de  I'lnst.  Pasteur,  viii.  853.  Quincke 
and  Stiihlen,  Berl.  klin.  Wchnschr.  (1894),  351.  A.  Fraenkel.  Centralbl. 
f.  klin.  Med.  (1886),  No.  10.  E.  Fraenkel  and  Simmonds,  ibid.  (1886),  No. 
39.  Achalrne,  Semaine  mtd.  (1890),  No.  27.  Grawitz,  Charite-Ann. 
xvii.  228.  Beumer  and  Peiper,  Centralbl.  f.  klin.  Med.  (1887),  No. 
4  ;  Ztechr.  f.  Hyg.  i.  489  ;  ii.  110,  382.  Sirotinin,  ibid.  i.  465.  R. 
Pfeiffer  and  Kolle,  Ztschr.  f.  Hyg.  xxi.  203.  R.  Pfeifter,  Deutsche  med. 
Wchnschr.  (1894),  898.  Sanarelli,  Ann.  de  I'lnst.  Pasteur,  vi.  721  ; 


BIBLIOGRAPHY  661 

viii.  193,  353.  Brieger  and  Fraenkel,  Bcrl.  klin.  Wchnschr.  (1890), 
241,  268.  Siduey  Martin,  Brit.  Med.  Journ.  (1898),  i.  1569,  1644  ;  ii. 
11,  73.  Bokenham,  Trans.  Path.  Soc.  London  (1898),  xlix.  373. 
Macta.lv. 'ii.  l>roc.  Roy.  Soc.  London,  B.  Ixxvii.  548.  Macfadyen  and 
Rowland,  Centralbl.  f.  Bakteriol.  u.  Parasitcnk.  (Orig. )  xxxiv.  618, 
765.  Lorrain  Smith  and  Tennant,  Brit.  Med.  Journ.  (1899),  i.  193. 
< 'astdlani,  Ztschr.  f.  Hyg.  u.  Infectionakrankh.  xl.  i. 

Ki'iiu-'.MioiAXJY  OF  TYPHOID.—  Forster  and  Kayser,  Munchen.  med. 
Wchnschr.  (1905),  4173.  Forster,  ibid.  (1908),  1.  Forster,  Discussion  at 
Unterelsiissischer  Artzverein,  Deutsche  med.  Wchnschr.  (1907),  85,  1767. 
Klinger,  Arb.  a.  d.  k.  gsndhtsamle.  (1906),  xxiv.  91.  Conradi  and 
their  authors,  Klin.  Jahrb.  (1907),  xvii.  115-433  ;  ibid.  (1909),  xxi.  171- 
-121.  (Typhoid  Carriers)  Dean,  Brit.  Med.  Journ.  (1908),  i.  562. 
Lcdingluiiii,  M.  and  J.  C.  G.,  ibid.  i.  15.  Sacquepee,  Bull,  de  I' List. 
J'ii*ti-nr,  viii.  1,  49  (with  literature).  Browning  and  Gilmour,  Glasgow 
.}/,,/.  Jnnrn..  (1910),  Ixxiv.  81. 

IMMUNITY  PHENOMENA  AND  SERUM  DIAGNOSIS. — Brieger,  Kitasato, 
and  Wassermanu,  Ztschr.  f.  Hyg.  xii.  137.  Widal,  Semainc  m<!d.  (1896), 
295,  303.  Achard,  ibid.  295,  303.  Griinbaum,  Lancet,  Sept.  1896., 
IMrj.iue,  Brit.  Med.  Journ.  (1897),  i.  529,  967  ;  Lancet,  Dec.  1896.' 
Remlinger  and  Schneider,  Ann.  de  Vlnst.  Pasteur,  xi.  55,  829.  Widal 
and  Sicard,  ibid.  xi.  353.  Peckham,  Journ.  Expcr.  Med.  ii.  549. 
Richardson,  ibid.  iii.  329.  Wright  and  Semple,  Brit.  Med.  Journ.  (1897), 
i.  256.  Wright  and  Lamb,  Lancet  (1899),  ii.  1727.  Wright,  ibid. 
(1900),  i.  150  ;  ii.  1556  ;  ibid.  (1901),  i.  609,  858,  1272,  1532  ;  ii.  715, 
1107  ;  ibid.  (1902),  ii.  651  ;  Brit.  Med.  Journ.  (1900),  ii.  113  ;  ibid. 
(1901),  i.  645,  771.  Wright  and  Leishman,  ibid.  (1900),  i.  622.  See 
also  discussion  at  the  0/.in.  Soc.  London,  Brit.  Med.  Journ.  (1901),  ii. 
1342.  Cha'nternesse  and  Widal,  Ann.  de  I'List.  Pasteur,  vi.  755. 
Christophers,  llrit.  M><>.  Journ.  (1898),  i.  71.  Remy,  Ann.  de.  Vlnst. 
Pitstein;  xiv.  55fi,  705.  Wyatt  Johnson,  Brit.  Med.  Journ.  (1897),  i.  231  ; 
Lancet  (1897),  ii.  1746.  Durham,  Lancet  (1898),  i.  154  ;  ibid.  ii.  446. 
(Vaccination  treatment  of  Typhoid  Fever)  Smallman,  Journ.  R.A.M.C. 
(1909),  vii.  136.  Leishman,  Journ.  Roy.  Intst.  Pub.  Health  (1910),  viii. 
385,  513. 

PARATYPHOID  AND  FooD-PoisoNix<;  BACILLI. — Boycott,  Journ.  Hyg. 
vi.  33.  Gaertner,  refs.  inde  Baumgarten's  Jahrcsbericht,  iv.  249  ;  vii. 
297  ;  xii.  508.  van  Ermengem,  in  Kolle  and  Wassermann's  Hnndbuch, 
vol.  ii.  Conradi,  Deutsche  Med.  Wchnschr.  (1904),  1165.  Foruet,  Arb. 
a.  i/.  /..  gtndktmmie.  (1907),  xxv.  247.  Levy  and  Gaehtgeus,  ibid.  xxv. 
250.  Goehtgeas,  Ibid.  (1909),  xxx.  610.  Rimpau,  ibid.  xxx.  330. 
llainhridge,  Journ.  of  Path,  and  Bacterial.  (1909),  xiii.  443.  Saoqn6pe£, 
Hull,  de  Vlnst.  Pasteur  (1907),  v.  888.  Sacquepee  and  Chevrel,  ibid. 
49,  97.  (Psittacosis)  Baumgartcn'.s  Jahrcsbericht.  xii.  496.  (Bacillus 
Enteritidis  Sporogenes)  Klein,  Rep.  Med.  Off.  Local  Govt.  Board,  xxv. 
171  ;  xxvii.  210. 

P.  A<  TI.IMAL  DYSENTERY.—  Shiga,  Centralbl.  f.  Bakteriol.  u.  Parasitenk. 
xxiii.  599  ;  xxiv.  817,  870,  913.  Kruse,  Deutsche  med.  Wchnschr. 
(1900),  637.  Flexner,  Bull.  Johns  Hopkins  Hasp.  (1900),  xi.  39,  231  ; 
Hfit.  .]/,,/.  Jnnrn.  (1900),  ii.  917.  Strong  and  Musgrave,  Journ.  Amcr. 
Med.  Assoc.  (1900),  xxxv.  498.  Vedder  and  Duval,  Journ.  Exper.  Med. 
(1902),  vi.  181.  Ogata,  Centralbl.  f.  Bakteriol.  u.  Parasitenk.  xi.  264. 
See  various  authors  in  Studies  from  the  Rockefeller  Institute  for  Medical 
ll<  search  (1904),  vol.  ii.  Park,  Collins,  and  Goodwin,  Journ.  Med. 


662  BIBLIOGRAPHY 

Research  (1904),  xi.  553.  Hiss,  ibid.  (1905),  xiii.  1.  Torrey,  Journ. 
fixper.  Med.  (1905),  vii.  365.  Weaver,  Tuniiiclifle,  Heiiiemaiin,  and 
Michael,  Journ.  Infect.  Diseases,  ii.  70.  Doerr,  Das  Dysenterietoxin, 
Jena,  1907  ;  Kraus  and  Levaditis'  Handbuch  (1908),  ii.  164.  Pane  and 
Lotte,  Centralbl.f.  fiaktcriol.  n.  Para  site n.k.  (Orig.j  (1907),  811.  Shiga, 
Ztschr.  f.  Hytj.  (1908),  Ix.  75.  Anmko,  ibid.  Ix.  85.  Franchietti,  ibid. 
Ix.  127.' 

SUMMER  DIAUUIKEA. — Morgan,  Brit.  Med.  Journ.  (1906),  i.  908  ; 
(1907),  ii.  16.  Morgan  and  Ledingham,  Proc.  Roy.  Soc.  Med.  (1909), 
ii.  (2)  (Epidcmiological  section),  133. 

OH  A  PTEK  XVI.  — Di  r  HT  1 1  v.  i:  i  A  . 


Klebs,  Vcrhandl.  d.  Cong.  f.  iiim-rc  Med.  (1883),  ii.  Loftier,  Mitth. 
a.  d.  k.  Gsndhtsamte.  (1884),  421.  Roux  and  Yersin,  Ann.  de  I'lnst. 
Pasteur,  ii.  629  ;  iii.  273  ;  iv.  385.  Brieger  and  Fraenkel,  fieri,  kiln. 
Wchnschr.  (1890),  241,  268.  Spronck,  Cenfralbl.  f.  ally.  Path.  u.  path. 
Anat.  i.  No.  25  ;  iii.  No.  1.  Welch  and  Abbott,  ^  Johns  Hopkin*  Hosp. 
Bull.  1891.  Reining  and  Wernicke,  Ztschr.  f.  Hyy.  xii.  10.  Loftier, 
Centralbl.  f.  fiaktcriol.  u.  Parasitcnk.  ii.  105.  v.  Hofmann.  Wicn. 
med.  Wchnschr.  (1888),  Nos.  3  and  4.  Cobbett  and  Phillips,  Journ. 
Path,  and  Barteriol.  iv.  193.  Peters,  ibid.  iv.  181.  Wright,  7,Wr,/, 
Med.  and  >S'.  Journ.  (1894),  329,  357.  Kanthack  and  Stephens,  Journ. 
Path,  and  Bacteriol.  iv.  45.  Klein,  Brit.  Med.  Journ.  (1894),  ii.  1393  ; 
(1895),  i.  100;  Hep.  Med.  O/.  Local  (fort.  Board  (1890-91 ),  219;  (1891- 
92),  125.  Abbott,  Journ.  Path,  and  Bacterial,  ii.  35.  Guinochet,  Com/it, 
rend.  $oc.  de  biol.  (1892),  480.  Roux  and  Martin,  Ann.  dr.  I'lnst. 
Pasteur,  viii.  609.  Cartwriglit  Wood,  Lancet  (1896),  i.  980,  1076  ; 
ii.  1145.  Sidney  Martin,  "  Goulstonian  Lectures,"  Brit.  Med.  Journ. 
(1892),  i.  641,  696,  755  ;  Rep.  Med.  Off'.  Loral  ffoct.  Board  ( 1891-92),  147  ; 
(1892-93),  427.  Escherich,  Wicn.  'med.  IVchnschr.  (1893),  Nos.  47-50  ; 
Wicn.  klin.  Wchnschr.  (1893),  Nos.  7-10;  (1894),  No.  22;  fieri,  klin. 
Wchnschr.  (1893),  Nos.  21,  22,  23.  Behring,  "Die  Geschichte  der 
Diphtherie,"  Leipzig,  1893;  "  Abhandlungen  /.  atiol.  Therap.  v.  anst. 
Krankh.,"  Leipzig,  1893;  ">Bckampfung  der  Infectionskrankheiteji," 
Leipzig,  1894.  Ehrlich  and  Wassermann,  Ztschr.  f.  llyg.  xviii.  23i». 


Deutsche  med.  Wchnschr.  (1894),  353.  Funck,  Ztschr.  f.  //)/</.  xvii.  401. 
Prochaska,  ibid.  xxiv.  373.  Madaen,  ibid.  xxiv.  425.  Neisser,  ibid. 
xxiv.  443  ;  Hyy.  Rundsch.  xiii.  705.  L.  Martin,  Ann.  dc.  Flnst. 
Pasteur,  xii.  26.  Park  and  Williams,  Journ.  E;qtcr.  Med.  i.  164. 
Salomonsen  and  Madsen,  ibid.  xii.  763.  Wood  head,  Brit.  Med.  Journ. 
(1898),  ii.  893  ;  Rep.  Metrop.  Asyl.  Bd.,  London,  1901.  Mi- tin,  Ann.  de 
I' List.  Pasteur,  xii.  596.  Madsen,  ibid.  xiii.  568,  801.  Dean  and  Todd, 
Journ.  of  Hyy.  ii.  194.  Cobbett,  ibid.  i.  485.  Graham-Smith,  ibid.  iv. 
258  ;  vi.  286.  Petrie,  ibid.  v.  134.  Hist,  Compt.  rend.  Moc.  de  biol. 
(1903),  No.  25.  Neisser,  fieri,  klin.  Wchnschr.  (1904),  No.  11.  Knapp, 
Journ.  Med.  Research  (1904),  475.  Morgenroth,  Ztschr.  f.  Hyg.  xlviii. 
177.  Bolton.  Lancet  (1905),  i.  1117.  Theobald  Smith,  Journ.  Med. 
Research  (1905),  xiii.  341.  Boycott,  Journ.  of  Hyg.  v.  223.  Ford 
Robertson,  Brit.  Med.  Journ.  (1903),  ii.  1065,  and  Rev.  of  Neurol.  and 
Psych,  vols.  i.-iii.  Nuttall  and  Graham-Smith,  "The  Bacteriology  of 
Diphtheria"  (with  full  literature,  etc.),  Cambridge,  1908. 


BIBLIOGRAPHY  663 


CHAPTER  XVII.—  TETANUS, 

Nicolaicr,  "  Beitriige  zur  Aetiologie  des  Wundstarrkrampfes,"  Inaug. 
Diss.  Giittingen,  1885.  Rosenbach,  Arch.  f.  kiln.  ('/«'>-.  xxxiv.  306. 
Carle  and  Rattoiie,  Gior.  <t.  r.  Accad.  di  med.  di  Torino,  1884.  Kitasato, 
Ztxch,:  f.  lhj<j.  vii.  225;  x.  267;  xii.  256.  Kitasato  an.  I  Weyl,  ibid. 
viii.  41,  404.  Vaillard,  Ann.  de  I'  lust.  Pasteur,  vi.  224,  676.  Vaillard 
and  Rouget,  ibid.  vi.  385.  Hehring,  "  Abhandlungcn.  z.  iitiol.  Therap. 
v.  anst.  Krankh.,"  Leipzig,  1893;  Ztschr.  f.  Hy<j.  xii.  1,45;  "  Blut- 
smimtherawe,"  Leipzig,  1892;  "  Das  Tetanusheilserum,"  Leipzig,  1892. 
Brii-ger  and  Fraenkel,  JJerl.  klin.  Wchnschr.  (1890),  241,  268.  Sidney 
Martin,  lleji.  Med.  Off.  Local  Go  vt.  Board  (1893-94),  497  ;  (1894-95),  505. 
Tsdiinsky,  Centralbl.  f.  Bakteriol.  u.  Parasitenk.  xiv.  316.  Tizzoni  ;md 
Cattani,  Arch.  f.  cxj>cr.  Path.  n.  Pharmakol.  xxvii.  432;  Centralbl.  f. 
Balctcriol.  u.  Parasitenk.  ix.  189,  685  ;  x.  33,  576  (Ref.)  ;  xi.  325  ;  Ji,-i-/. 
kiln.  U'chiixrhr.  (1894),  732.  Madsen,  Ztschr.  f.  Hyg.  xxxii.  214.  Ritchie, 
./'>n  ni.  of  Jfit'f.  i.  125.  Danysz,  Ann.  de  Vlnst.  Pasteur,  xiii.  155. 
Marie  and  Morax,  Ann.  dc  Vlnst.  Pasteur,  Paris,  xvi.  818.  Meyer  and 
Ransom,  Proc.  Roy.  Soc.  London,  Ixxii.  26  ;  Arch.  /.  cxper.  Path.  n. 
riinrmukol.,  Leipzig,  xlix.  269.  Roux  and  Borrel,  Ann.  dc  Vlnst.  Pasteur, 
Paris,  xii.  225.  Henderson  Smith,  Journ.  Hyy.  vii.  205.  Kitt,  see  ref.  in 
('••nh-albl.  f.  Bakteriol.  u.  Parasitenk.,  Referate,  xxxii.  359.  Eisler  and 
I'ribram  in  Krans  and  Levaditi's  Hantlbuch,  i.  103. 

MALK;NANT(KIH..MA.—  Pasteur,  Bull.  Acad.  denied.,  1881,  1887.  Koch, 
Mitt/i.  ".  'I.  /.-.  ttxmUiisamtc.  i.  54.  Kitt,  -A////r.s/>.  '/.  /,-.  <'<'nlr.-  7'/n'<  ,-«  /-./"<- 
s,-h>'/>  In  Mihich'',!,  1883-84.  W.  R.  Hesse,  Deutsche  med.  Wchnschr. 
(1885),  No.  14.  Chauveau  and  Arloing,  Arch.  vtt.  (1884),  366,  817. 
Liborius,  Zfsrit,:  f.  //////.  i.  115.  Roux  and  Chamberland,  Ann,,  tie  Vlnst. 
,  i.  ;")t;-J.  Charriii  and  Roger,  Couijrt.  rent/.  Soc.  dc  bio/.  (1877),  st'-r. 


viii.  vol.  iv.  ]>.  408.  Kerry  and  S.  Fraenkel,  Ztschr.  /.  Ify</.  xii.  204. 
Sanfelice,  -ibid.  xiv.  339.  Leelainche  and  Velle,  Ann.  dc  Vlnst.  Pasteur, 
xiv.  202,  590. 

BACILM-S  PxnTLixrs.—  v.  Ermengeni,  Ccntralbl.  f.  Bakteriol.  ». 
Pui-iixiteiik.  xix.  443;  Ztschr.  f.  Hyy.  xxvi.  1.  Kempner,  ibid.  xxvi.  481. 
Kcnipner  and  Schepilewsky.  ibid,  xxvii.  213.  Kempner  and  Pollack, 
It.-,!/*-/,,-  med.  Wchiischr.  (1897),  No.  32.  Brieger  and  Keinpner,  ibid. 
(1897),  No.  33.  Marinesco,  Compt.  rend.  Soc.  dc  biol.  (1896),  No.  31. 
Schneidemiihl,  Centralbl.  f.  Bakteriol.  u.  Parasitenk.  xxiv.  577,  619. 
R.'inrr.  ibid,  xxvii.  857.  Madsen  in  Kraus  and  LevinlitVsHaiidbtrch,  i.  137  ; 
ii.  134.  Leuchs,  Ztschr.  f.  Hijy.  u.  Infektionskrankh.  (1910),  Ixv.  55. 

QuAUTKi:-K\  IL.—  See  Nocard  and  Leclainche,  "Les  maladies  micro- 
l>ieniics  dcs  aninianx,"  Paris,  1896.  Arloing  Cornevin,  et  Thomas, 
"  Le  charbon  syniptomatiqne  du  brenf,"  Paris,  1887.  Nocard  and  Roux, 
./////.  ill-  r/nsf.  PasUur,  i.  256.  Roux,  ibid.  ii.  49.  See  also  Joi'/-». 
<'»,,ir.  /'nt/i.  u,  ,,l  Therap.  iii.  253,  346  ;  viii.  166,  233.  Grassberger  and 
Schattentroh  in  Kraus  and  Levaditi's  Handbuch,  i.  161  ;  ii.  186.  Eism- 
berg,  Comp.  rend.  Soc.  dc  biol.  No.  62,  491,  537,  613. 

BACILLUS  AEKOGRKES  CAPSULATUS.  —  Welch  and  Nuttall,  Bull.  Johns 
Hopkins  Il»*i>.  (1892),  81.     Welch  and  Flexner,  Journ.  Expcr.   Med.  i. 
5.     E.  Fraenkel,  Centralbl.  f.  Balteriol.  n.  Parasitenk.  xiii.  13.     Durham, 
Hulf.  Johns  Hopkins  Hosp.  (1897),   68.     Norris,  Am.  Journ.  Med. 
cxvii.  172. 

I'l  siFOKM  BACILLI.—  Babes  in  Kolle  and  Wassj-rmann's  Handbuch, 
Erg;inz-Bd.  i.  271.  Vincent,  Ann.  dc  l'J,>sf.  p.isteur  (1896),  x.  492-; 


664  BIBLIOGRAPHY 

(1899),  xiii.  609.  Areillon  and  Zuber,  Arch,  de  med.  exper.  (1898),  x. 
517.  Bernheim,  Centralbl.  f.  Bakteriol.  u.  Parasitenk.  (1898),  xxiii.  171. 
Plant,  Deutsche,  med.  Wchnschr.  (1904),  920.  Beitzke,  Centralbl.  f. 
Bakteriol.  u.  Parasitenk.  (Ref.)  (1904),  xxxv.  1.  Ellermann,  ibid.  (Orig.) 
(1904),  xxxvii.  729  ;  xxxviii.  383  ;  Ztschr.  f.  Hyg.  (1907),  Ivi.  453. 
Veszpremi,  Centralbl.  f.  Bakteriol.  u.  Parasitenk.  (Orig.)  xxxviii.  136. 
Weaver  and  Tunnicliffe,  Journ.  Infect.  Diseases,  (1905),  ii.  446  ;  (1906), 
iii.  190.  Blumer  and  MacFarlane,  Amer.  Journ.  Med.  Sc.  clxl.  122. 


CHAPTER  XVIII.— CHOLERA. 

Koch,  Rep.  of  1st  Cholera  Conference,  1884  (v.  "  Microparasites  in 
Disease, "New  Sydenham  8oc.,  1886).  Nikuti  and  Rietsch,  Compt.  rend. 
Acad.  d.  sc.  xcix.  928, 1145.  Bosk,  Ann.  de  I'lnst.  Pasteur,  ix.  507.  Petten- 
kofer,  Munchen.  med.  Wchnschr.  (1892),  No.  46  ;  (1894),  No.  10.  Sawts- 
clienko,  Centralbl.  f.  Bakteriol.  u.  Parasitenk.  xii.  893.  Pfeiffer,  Ztschr.  f. 
Hyg.xi.393.  Kolle.^'oJ.  xvi.  329.  Issaeff  and  Kolle,  ibid,  xviii.  17.  Wasser- 
mann,  ibid.  xiv.  35.  Soberuheini,  ibid.  xiv.  485.  Metchnikoff,  Ann.  de 
I'lnst.  Pasteur,  vii.  403,  562  ;  viii.  257,  529.  Fraenkel  and  Sobefnheim, 
Hyg.  Rundschau,  iv.  97.  Dunbar,  Arb.  a.  d.  k.  Gmdhtsamte.  ix.  379. 
Pf'eiffer  and  Wasserman,  Ztschr.  f.  Hyg.  xiv.  46.  Wesbrook,  Ann.  de 
I'lnst.  Pasteur,  viii.  318.  Scholl,  Berl.  klin.  Wchnschr.  (1890),  No.  41. 
Griiber  and  Wiener,  Arch.  f.  Hyg.  xv.  241.  Cunningham,  Sclent.  M em. 
Med.  Off.  India,  1890  and  1894.  Hueppe,  Deutsche  med.  Wchnschr. 
(1889),  No.  33.  Klemperer,  ibid.  (1894),  435  ;  Berl.  klin.  Wchnschr. 
(1892),  969.  Lazarus,  ibid.  (1892),  1071.  Reincke,  Deutsche  med.  Wchnschr. 
(1894),  795.  Koch,  Ztschr.  f.  Hyg.  xiv.  319.  Voges,  Centralbl.  f. 
Bakteriol.  u.  Parasitenk.  xv.  453.  Pastana  and  Bettencourt,  Centralbl. 
f.  Bakteriol.  u.  Parasitenk.  xvi.  401.  Dieudonne,  ibid.  xiv.  323.  Celli 
and  Santori,  ibid.  xv.  289.  Neisser,  ibid.  xiv.  666.  Sanarelli,  Ann.  de 
I'lnst.  Pasteur,  vii.  693.  Ivanoff,  Ztschr.  f.  Hyg.  xv.  485.  Issaeff,  ibid. 
xvi.  286.  Pfuhl,  ibid.  x.  510.  Rurnpel,  Deutsche  med.  Wchnschr.  (1893), 
160.  Klein,  Rep.  Med.  Off.  Local  Govt.  Board,  1893:  "Micro-organisms 
and  Disease,"  London,  1896.  Haffkine,  Brit.  Med.  Journ.  (1895),  ii. 
1541;  Indian  Med.  Gaz.  (1895),  No.  1  ;  "Anti-cholera  Inoculation," 
Rep.  San.  Com.  India,  Calcutta,  1895.  Pfeiffer  in  Fliigge,  "Die  Micro- 
organismen,"  3rd  ed.  1896.  Gamaleia,  Ann.  de  I'lnst.  Pasteur,  ii.  482, 
552.  Archard  and  Bensande,  Semaine  med.  (1897),  151.  Rumpf,  "Die 
Cholera  Asiatica  und  Nostras,"  Jena,  1898.  Kraus  and  Pribram,  Centralbl. 
f.  Bakteriol.  xli.  (Orig.),  15,  155.  Kraus  and  Prantschoff,  ibid.  377,  480. 
A.  Macfadyen,  ibid.  xlii.  (Orig.),  365.  Gotschlich,  Scientific  Reps. 
Sanitary,  Maritime,  and  Quarantine  Council  of  Egypt,  Alexandria,  1905, 
1906.  For  discussion,  vide  Supplements  to  Centralbl.  f.  Bakteriol.  (Ref. ), 
(1906),  xxxviii.  84  ;  and  (1908),  xlii.  1.  Dunbar,  Berlin,  klin.  Wchnschr. 
(1902),  No.  39.  Kraus,  in  Kraus  and  Levaditi's  "  Handbuch  der 
Immunitatsforschung  (with  literature  on  toxins  and  anti-toxins).  For 
Russian  epidemic,  vide  Centralbl.  f.  Bakteriol.  u.  Parasitenk.  (Ref.) 
(1909),  xliv.  1  et  seq.  ;  Dieudonne,  Centralbl.  f.  Bakteriol.  u.  Parasitenk. 
(Orig.)l.  107. 

CHAPTER  XIX. — INFLUENZA,  ETC. 

INFLUENZA. — Pfeiffer,  Kitasato,  and  Canon,  Deutsche  med.  Wchnschr. 
xviii.  28,  and  Brit.  Med.  Journ.  (1892),  i.  128.  Babes,  Deutsche  med. 


BIBLIOGRAPHY  665 

r.  xviii.  113.  Pfeiffer  and  Beck,  ibid.  (1892),  465.  Pfuhl, 
C<-,,tr«lbl.  f.  Bakteriol.  n.  Parasitenk.  xi.  397.  Klein,  Rep.  Med.  Off. 
/,<«•»//  <;,,,-/.  /,w.>-^(1893),  85.  Pfeiffer,  Ztschr.  f.  Hyg.  xiii.  357.  Huber, 
yjwh,:  f.  ////</.  xv.  454.  Kruse,  Deutsche  med.  Wchnschr.  (1894),  513. 
Pelicke,  BerL  kiln.  Wchnschr.  (1894),  524.  Pfuhl  and  Walter,  Devtsclt,- 
ini'il.  Wchnschr.  (1896),  82,  105.  Cantani,  Ztschr.  f.  Hyg.  xxiii.  265. 
I'lulil,  Ztschr.  f.  Hyg.  xxvi.  112.  Wasserniann,  Deutsche  med.  Wchnwltr. 
(1900),  No.  28.  Clemens,  Miinchen.  med.  Wchmchr.  (1900),  No.  -11. 
\\\ n.,  ..op,  Journ.  Med.  Ass.,  February  1903.  Neisser,  Deutsche  med. 
//V////.sr/i/\  (1903),  No.  26.  Auerbach,  Ztschr.  f.  Hyg.  (1904),  xlviii.  259. 
ini,  Centralbl.f.  Bakteriol.  u.  Parasitenk,  (Orig.)  (1907),  xliii.  407. 
r,  Ibid.  (Orig. )  (1909),  1.  503. 

Nu-CouGH. — Jochmann,  Arch.  f.  klin.  Med.  Ixxxiv.  470. 
and  Krause,  Ztschr.  f.  Hyg.  (1901),  xxxvi.  193.  Sperigler, 
Deutsche  med.  Wchnschr.  (1897),  830.  Davis,  Journ.  Infect.  Diseases,  iii.  1. 
Bordet  and  Gengou,  Ann.  de  I'lnst.  Pasteur,  xx.  731  ;  xxi.  720  ; 
<  ',„(,•'•  H./.f.  Bakteriol.  u.  Parasitenk.  (1909)  (Ref. ),  xliii.  273.  Bordet,  Bull, 
de  1'Acad.  Jioy.  de  Medicine  de  Belgique  (1908),  4th  ser.  tome  xxii.  729. 
Arnhuini,  Berlin,  klin.  Wchnschr.  (1908),  1453.  Fraenkel,  Miinchen.  med. 
Wchnschr.  (1908),  1683.  Klimenko,  Centralbl.f.  Bakteriol.  u.  Parasitenk.. 
(Orig.)  xlviii!  64.  Wollstein,  Journ.  Exper.  Med.  (1909),  xi.  41. 

PLAGUE. — Kitasato,  Lancet  (1894),  ii.  428.  Yersin,  Ann.  de  I'lnst. 
J'nsti'in;  viii.  6»!2.  Lowson,  Lancet  (1895),  ii.  199.  Yersin,  Calmctte, 
:ind  l>orrel,  Ann.  de  I'lnst.  Pasteur,  ix.  589.  Aoyama,  Centralbl.  f. 
Hnklr.riol.  u.  Parasitenk.  xix.  481.  Zettnow,  Ztschr.  f.  Hjig.  xxi.  164. 
Yersin,  Ann.  de  I'lnst.  Pasteur,  xi.  81.  Gordon,  Lancet  (1899),  i.  688. 
Simond,  Ann.  de  VInst.  Pasteur,  xii.  625.  Haflkine,  Brit.  Med.  Journ. 
(1S97),  i.  424.  Wyssokowitz  and  Zabolotny,  Ann.  de  I'lnst.  Pasteur, 
xi.  663.  Ogata,  Centralbl.f.  Bakteriol.  u.  Parasitenk.  xxi.  769.  Childe, 
llfit.  Med.  Journ.  (1898),  ii.  858.  See  also  Brit.  Med.  Journ.  and 
Lancet,  1897-99.  Ltistigand  Galeotti,  Deutsche  med.  Wchnschr.  (1897), 
No.  15.  Markl,  Centralbl.f.  Bakteriol.  u.  Parasitenk.  xxiv.  641,  728; 
xxix.  810.  Cairns,  Lancet  (1901),  i.  1746.  Montenegro,  "Bubonic 
Pl.-igue,"  London,  1900.  Netter,  "  La  peste  et  son  bacillc,"  Paris,  1900. 
Mitth.  d«T  Deutschen  Pest-Kommission,  Deutsche  med.  Wchnschr.  (1897), 
Nos.  17,  19,  31,  32.  "Report  of  the  India  Plague  Commission  (1898- 
99),"  London,  1900-1901.  Also  numerous  papers  in  the  Lancet  and  Brit. 
M><l.  Jowr*.t  1897-1901.  Regarding  Glasgow  epidemic,  see  ibid.  (1900), 
ii.  "Reports  on  Plague  Investigations  in  India,"  Journ.  Hyg.  (1906), 
vi.  422;  (1907),  vii.  323;  (1908),  viii.  162.  Lamb,  "The  Etiology 
;in«l  Epidemiology  of  Plague,"  Calcutta,  1908.  Lfston,  Report  Bombay 
J!»<-/.  /,////.  (1908),  ii. 

MALTV  KKVKK -Bruce,  Practitioner,  xxxix.  160;  xl.  241;  Ann.  de 
/'/us/.  r«*t',ir,  vii.  291.  Bruce,  Hughes,  and  Westcott,  Brit.  Med. 
Journ.  (1887),  ii.  58.  Hughes,  Ann.  de.  I'lnst.  Pasteur,  vii.  628  ;  Lancet 
(1892),  ii.  1265.  Wright  and  Semple,  Brit.  Med.  Journ.  (1897),  i.  1214. 
Wright  and  Smith,  ibid.  (1897),  i.  911  ;  Lancet  (1897),  i.  656.  Welch, 
ihiil.  (1897),  i.  1512.  Gordon,  ibid.  (1899),  i.  688.  Durham,  Journ. 
/'iitli.  a  ad  Bacteriol.  v.  377.  Bruce  in  Davidson's  "  Hygiene  and 
Diseases  of  Warm  Climates,"  Edinburgh  and  London,  1893.  Birt  and 
Lamb,  Lancet  (1899),  ii.  701.  Brunner,  Wien.  klin.  Wchnschr.  (1900), 
No.  7.  Bruce,  Journ.  Roy.  Army  Med.  Corps  (1904),  ii.  487,  731  ; 
(1907),  viii.  225.  Horrocks,  Proc.  Roy.  Soc.  London,  Series  B  (1905), 
Ixxvi.  510.  "Reports  of  the  Commission  on  Mediterranean  Fever," 


666  BIBLIOGRAPHY 

1904-1907  (reprinted  in  Journ.  Roy.  Army  Med.  Corps,}.  Eyre  in  Kolle 
and  Wassermann's  Handbuch  d.  Patho<j.  Mikro-orr/anismen,  Eryaiizunys- 
band,  1906.  Milroy,  "Lectures  on  Militensis  Septicaimia,"  Lancet  (1908), 
i.  1677,  et  seq.  Sergent,  Gillot,  et  Lemaire,  Ann.  de  Vlnst.  Pasteur,  xxii. 
209.  Siere,  ibid.  xxii.  616. 

CHAPTER   XX.—  DISEASES  DUE  TO  SPIROCILKTES. 

RELAPSING  FEVERS. — Obermeier,  Centralbl.  f.  d.  med.  Wissensch. 
(1873),  145;  and  Berl.  klin.  Wcknschr.  (1873),  No.  35.  Munch, 
Centralbl.  f.  d.  med.  Wissensch.,  1876.  Kocli,  Deutsche  med.  Wchnschr. 
(1879),  327.  Moczutkowsky,  Deutsches  Arch.  f.  klin.  Med.  xxiv.  192. 
Vandyke  Carter,  Med.-Chir.  Trans.,  London  (1880),  78.  Lubinoff, 
Virchow's  Archiv,  xcviii.  160.  Metchnikolf,  ibid.  cix.  176.  Soudake- 
witch,  Ann.  dc  I  Inst.  Pasteur,  v.  545.  Lamb,  Sclent.  Mem.  Med.  Off. 
India  (1901),  pt.  xii.  77.  Sawtschenko  and  Melkich,  Ann.  de  VInxt. 
Pasteur,  xv.  497.  Tictin,  Centralbl.  f.  Bakteriol.  xxi.  179.  Karlinski, 
Centralbl.  f.  Baktcriol.  (1902)  (Orig.)  xxxi.  566.  Gabritschewsky,  Ztschr. 
f.  klin.  Med.  (1905),  Bd.  56.  Norris,  Pappenheimer,  Flournoy,  Journ. 
Infect.  Diseases,  iii.  266.  Novyand  Knapp,  ibid.  291.  Zettnow,  Ztschr.  f. 
Hyg.  (1906),  Hi.  485;  Deutsche  med.  Wchnschr.,  1906.  Maiiteufel,  Arl. 
a.  d.  k.  Gsndhtsamte.  xxix.  337.  Shellack,  ibid.  xxx.  351.  Novy, 
Journ.  Amer.  Med.  Assoc  xlvii.  215.  Mackie,  Lancet  (1907),  ii.  832  ; 
Brit.  Med.  Journ.  (1907),  ii.  1706  ;  New  York  Med.  Jonni..,  Aug.  22, 
1908.  Strong,  Philippine  Journ.  Med.  Sc.  iv.  187. 

AFRICAN  TICK  FEVER. — Ross  and  Milne,  Brit.  Med.  Journ.  (1904), 
ii.  1453.  Dutton  and  Todd,  Thompson- Yules  Laboratory  Rep.  (1905),  vi. 
pt.  ii.  Koch,  Deutsche  mr.d.  JTchnschr.,  1905;  Berl.  klin.  Wchnschr., 
1906.  Hodges  and  Ross,  Brit.  Med.  Journ.  (1905),  i.  713.  Breuil  and 
Kinghorn,  ibid.  i.  668.  Bivuil,  Lancet  (1906),  i.  1806.  Levaditi,  Comjtt. 
Acad.  Sc.  (1906),  tome  142,  1099.  Leishman,  Journ.  R.A.M.C.  (1909), 
xii.  123.  Levaditi  and  Manom'lian,  Ann.  dc  Vlnst.  Pasteur  (1907), 
xxi.  205. 

SYPHILIS. — Lustgarten,  Wicn.  med.  Wchnschr.  (1884),  No.  47. 
Sabouraud,  Ann.  de  Vlnst.  Pasteur,  vi.  184.  Golas/,,  Journ.  d.  maJ. 
cutan.  et  syph.  (1894),  170.  Markuse,  Vrtljschr.  f.  Jkrmat.  u.  Sijph. 
(1883),  No.  3.  van  N lessen,  Centralbl.  f.  Bakteriol.  n.  Parasitcnk. 
xxiii.  49.  Metchnikoff  and  Roux,  Ann.  de  V Inst.  Pasteur,  xvii.-xix. 
Lassar,  Berl.  klin.  Wchnschr.  (1903),  1189.  Neisser,  Deutsche  med. 
Wchnschr.  (1904),  1369,  1431.  Schaudiun  and  Hoffmann,  Arb.  a.  d. 
k.  Gsndhtsamte.  (1905),  Bd.  22  ;  Deutsche  med.  Wchnxclir.  (1905), 
No.  18;  Berl.  klin.  Wchnschr.  (1905),  Nos.  22,  23.  Schaudinn, 
Deutsche  med.  Wchnschr.  (1905),  No.  22.  Hoffmann,  Berl.  klin. 
Wchnschr.  (1905),  No.  46.  "Selected  Essays  on  Syphilis  and  Smallpox," 
New  Sydenham  Society,  1906.  Metchnikoff,  La  Semaine  med.  (1905), 
234.  Levaditi,  ibid.  (1905),  247.  Siegel,  Miinchen.  med.  Wchnschr. 
(1905),  1321,  1384.  Herxheimer,  ibid.  (1905),  1857.  Shennan,  Lancet 
(1906),  i.  6b3,  746.  Maclennan,  Brit.  Med.  Journ.  (1906),  i.  1090. 
Levaditi,  Ann.  de  I'Inst.  Pasteur  (1906),  xx.  41.  Levaditi  and  M'Intosh, 
ibid.  (1907),  784.  Levaditi  and  Yamanouchi,  ibid.  (1908),  763.  Hoff- 
mann, "Die  Atiologie  der  Syphilis,"  Berlin,  1906.  Neisser,  "Die 
experimentelle  Syphilisforschung,"  Berlin,  1906. 

SERUM  DIAGNOSIS. — "Wassermann,  Neisser,  and  Bruck,  Deutsche  med. 
Wchnschr;  (1906),  745.  Wassermaun  and  Plant,  ibid.  1769.  Wassermann, 


BIBLIOGRAPHY  067 

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rilAPTKK    XXI.     IM.MI-MTV. 

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OPSONINS. — Denys  and  Leclef,  "La  cellule,"  1895,  177.  Sawtschenko, 
Ann.  de  V Inst.  Pasteur  (1902),  106.  Wright  and  Douglas,  Proc.  Roy. 
Soc.  London,  Ixxii.  357  ;  Ixxiii.  128  ;  Ixxiv.  147.  Wright  and  Reid,  ibid. 
Ixxvii.  211.  Bulloch  and  Atkin,  ibid.  Ixxiv.  379.  Dean,  ibid.  Ixxvi. 
506  ;  Brit.  Med.  Journ.  (1907),  ii.  1409.  Discussion  in  Centralbl.  f. 
Bakteriol.  u.  Parasitenk.  Referate  xliv.  Supplement  14.*  Bulloch  and 
Western,  Proc.  Roy.  Soc.  London,  Ixxvii.  531.  Neufeld  and  Rimpau, 
Deutsche  med.  Wchnschr.  (1904),  1458.  Neufeld,  Berl.  klin.  Wchnschr. 
(1908),  No.  21  :  Med.  Klinik.  (1908),  No.  19.  Hektoen  and  Ruediger, 
Journ.  Infect.  Diseases  (1905),  128.  Hektoen,  ibid.  (1908),  259  ;  (1909), 
78.  Leishman,  Trans.  Path.  Soc.  Lond.,  1905.  Muir  and  Martin,  Brit. 
Med.  Journ.  (1906),  ii.  ;  Proc.  Roy.  Soc.  London,  B.,  Ixxix.  187. 
Fornet  and  Porter,  Centralbl.f.  Bakteriol.  u.  Parasitenk.  (Orig.)  (1908), 
xlviii.  461. 

The  following  works  dealing  with  the  subject  of  Immunity  have  been 
published  within  recent  years: — Metchnikoff,  "  Immunity  in  Infective 
Diseases"  (Engl.  TransL),  Cambridge,  1905  ;  Ehrlich,  "Studies  in  Im- 
munity" (Engl.  Transl.),  2nd  ed.,  New  York,  1909  ;  Bordet,  "Studies  in 
Immunity,"  New  York,  1909;  Kraus  and  Levaditi,  "Handbuch  der 
Technik  und  Methodik  der  Immunitatsforschung,"  Jena,  1908  ;  Wright, 
"  Studies  on  Immunisation,"  London,  1909.  D'Este  Emery,  "  Immunity 
and  Specific  Therapy,"  London,  1909;  Muir,  "Studies  on  Immunity," 
London,  1909;  Wolff-Eisner,  "  Klinische  Immunitiitslehre  und  Serodiag- 


BIBLIOGRAPHY  669 

nostik,"  Jena,  1910.  The  most  important  papers  dealing  with  current 
work  on  the  subject  are  published  in  the  Zeitschrift  fur  Immunitdts- 
forschung. 

ANAPHYLAXIS.  —  Richet,  Compt.  rend.  Soc.  de  biol.,  1903-5,  Ann.  de 
r/nxt..  I'«xt..',ir  (1907),  xxi.  497  ;  (1908),  xxii.  465.  Arthus,  Compt.  rend. 
Soc.  de  biol.  (1903),  Iv.  817.  Arthus  and  Breton,  ibid.  Iv.  1478.  Th. 
Smith,  Discussion  on  "  Hypersensibility,"  in  Journ.  Amer.  Mcd.  Assoc. 
(1906),  xlvii.  1010.  Rosenau  and  Anderson,  Hya.  Lab.  Bull.,  Washington, 
Nos.  29,  39,  45  ;  Journ.  Infect.  Diseases  (1907),  vol.  iv.  1.  Otto,  in  v. 
Lriithold-Gedenkschrift,  Bd.i.  art.  "  Anaphylaxie/'inKolle-Wassermann's 
"  Handbuch,"  Ergiinz.-Bd.  ii.  Hft.  2.  Gay  and  Southard,  various  papers 
iu  Journ.  M«l.  Itixearch  (1907),  xvi.  et  seq.  Doerr,  art.  "  Anaphylaxie," 
in  Kraus-Lcvaditi's  "  Haudbuch."  Various  papers  by  Besredka  in  Ann.  de 
r  Inxt.  I'ltxt.-m:  1907.  i'f  wq.,  and  by  Biedl  and  Kraus,  Friedberger,  Doerr 


and  Russ,  in  Ztschr.  f.  Jmmnnitdtsf.  Bd.  ii.  et  seq.  Bail,  ibid.  (1909), 
iv.  470.  v.  Pirquet  and  Schick,  "Die  Seiumkrankheit,"  Wien,  1907. 
Currie,  Journ.  Hy<j.  (1907),  vii.  35,  61.  Goodall,  ibid.  607.  Scott, 
./mini.  I'nih.  mid  Bacterial.  (1909),  xiv.  147  and  (1910),  xv.  31.  Auer 
and  Li-wis,  Journ.  Amer.  Med.  Assoc.  (1909),  liii.  458. 

APPENDIX  A.—  SMALLPOX. 

Jenner,  "An  Inquiry  into  the  Causes  and  Effects  of  the  Variola 
Vaccime,"  London,  1798.  Creighton,  art.  "Vaccination"  in  Ency. 
I'.rlt.,  9th  cd.  Crookshank,  "Bacteriology  and  Infective  Diseases." 
M'Vail,  "Vaccination  Vindicated."  Chauveau,  Viennois  et  Mairet, 
'•  Vaccine  et  variole,  nouvelle  e"tude  experimentale  sur  la  question  de 
ridentitc-  de  res  deux  affections,"  Paris,  1865.  Klien,  Jb-p.  Med.  Off. 
Loot/  &ovt.  Hoard  (1892-93),  391  ;  (1893-94),  493.  Copeman,  Brit.  Med. 
Jour*.  (1894),  ii.  631  ;  Journ.  Path,  and  Bacterial,  ii.  407  ;  art.  in 
Clifford  Alllmtt's  "System  of  Medicine,"  vol.  ii.  L.  Pfeiffer,  "Die 
I'roto/oi-n  als  Krankheitserreger,"  Jena,  1891.  Ruffer,  Brit.  Med.  Jotmi. 
(1894),  June  30.  Becli-re,  Chambon,  and  Meiiard,  Ann.  de  VInst.  Pasteur, 
\.  1  ;  xii.  837.  Copeman,  "Vaccination,"  London,  1899.  Calmette  and 
Hiu'riii.  Ann.  dr.  V  Inst.  Pasteur,  xv.  161.  Guarnieri,  Centralbl.  f. 
l'»ikt'  r'ml.  u.  /'araffitenk.  xvi.  299.  Ewing,  Journ.  Med.  Research,  xiii. 
'!'•'>'.).  Pi'owa/ck,  Arb.  «.  <l.  kaiscrl.  Ge.sumllteitsamte,  xxii.  535  ;  xxiii. 
.".-jr..  \V;i<irlc\\ski,  Z/si-Jir.  f.  Hyg.  xxxviii.  212.  Bonhoff,  Berl.  /•////. 
//V////.sr/«/\  (I'.'O'.X  ]..  11-12.  Carini,  CwtiralW.  f.  BaJct<>rioL  u.  Pmrasttcrik. 
(Orig.)  xxxix.  6S5. 

APPENDIX  B.—  HVDUOPHOIHA. 


Pastoar,  r,t,n^.  revd.  Acad.  d.  sc.  xdi.  1259;  xcv.  1187;  xcviii.  lf,7, 
1229  ;  ci.  765  ;  cii.  459,  835  ;  ciii.  777.  Schaffer,  Ann.  de.  VInst.  Pant  fin: 
iii.  644.  Fit-mill^.  Trims.  1th  Internal.  Cony.  Hyg.  and  Demo;/,  iii.  16. 
Hdinan,  Ann.  de  I'  List.  Pasteur,  ii.  274  ;  iii.  15.  Babes  and  Lepp,  ibid. 
iii.  :JS-I.  Xocard  and  Roux,  ibid.  ii.  341.  Roux,  ibid.  i.  87  ;  ii.  479. 
Bruschettini.  <:,;i/,-albl.  f.  Bakteriol.  it.  Parasitenk.  xx.  214  ;  xxi.  203. 
Mi-mum,  ii'iit.  \\.  209  ;  xxi.  657.  Frantzins,  ibid,  xxiii.  782  ;  xxiv.  971. 
K.Mi.liimiT.  Ann.  /A-  n,<*i.  Pasteur,  xvii.  834;  xviii.  150;  xix.  «;2.'.. 
Harv.-y  :md  M.-K.-ndrick,  Sc.  Mem.  by  Officers  of  Med.  a<t>'  San  it.  />•/•'*. 
n'ort.  huiia  (New  Series),  No.  30  (1907),  Calcutta.  L-mm  and  M.-Ki-n- 
dri.-k.  ibid.  (1909),  No.  3(5.  Hiigyes,  Lyssa,  in  Nothnagers  "SjM-c.  Path. 
u.  Th.  T,"  Vienna,  1897. 


670  BIBLIOGFxAPHY 

NEGRI  BODIES. — Negri,  Ztschr.  f.  Hyg.  u.  Infcctionskrankh.  xliii. 
507  ;  xliv.  519  ;  Ixiii.  421.  Williams  and  Lowden,  Journ.  Inf.  Diseases, 
iii.  452.  Bertarelli,  Centralbl.  f.  Baktcriol.  xxvii.  556.  D'Amato  and 
Faggella,  Ztschr.  f.  Hyg.  (1910),  Ivi.  351.  Frosch,  in  Kolle  and 
Wassermann's  "Handbuch  der  Patliogenen  Mikro-organismen,"  Erganx- 
ungsband,  i.  626.  Frothingham,  Am.  Journ.  Pub.  Hyg.  (1908),  xviii. 

APPENDIX  C. — MALARIAL  FKVEK. 

Laveran,  Bull.  Acad.  de.  med.  (1880),  ser.  ii.  vol.  ix.  1346;  "  Du 
paludisme  et  de  son  hematozoaire,"  Paris,  1891.  Marchiafava  and  Celli, 
Fortschr.  d.  Med.,  1883  and  1885  ;  also  in  Virchow's  Festschrift.  Golgi, 
Arch.  per.  le.  sc.  med.,  1886  and  1889  ;  Fortschr.  d.  Med.  (1889),  No.  3  ; 
Ztschr.  f.  Hyg.  x.  136  ;  Deutsche  med.  Wclmschr.  (1892),  663,  685,  707, 
729.  Steinberg,  New  York  Med.  Rec.  xxix.  No.  18.  James,  ibid,  xxxiii. 
No.  10.  Councilman,  Fortschr.  d.  Med.  (1888),  Nos.  12,  13.  Osier, 
Trans.  Path.  8oc.  Philadelphia,  xii.  xiii.  Grass!  and  Feletti,  Riforma 
med.  (1890),  ii.  No.  50.  Carialis, ,  Fortschr.  d.  Med.  (1890),  Nos.  8,  9. 
Danilewsky,  Ann.  dc  I'lnst.  Pasteur,  xi.  758.  "Parasites  of  Malarial 
Fevers,"  New.  Syd.  Soc.,  1894  (Monographs  by  Marchiafava  and 
Bignami,  and  by  Mannaberg,  with  Bibliography).  Manson,  Brit.  Med. 
Journ.  (1894).  i.  1252,  1307  ;  Lancet  (1895),  ii.  302  ;  Bril.  Med.  Journ. 
(1898),  ii.  849  ;  Koch,  JBerl.  klin.  Wchnschr.  (1899),  69.  Ross,  Indian 
Med.  Gas.  xxxiii.  14,  133,  401,  448.  Nuttall,  Centralbl.  f.  Bakteriol.  u. 
Parasitenk.  xxv.  877,  903;  xxvi.  140;  xxvii.  193,  218,  260,  328  (with 
full  literature).  Manson,  Lancet  (1900),  i.  1417  ;  (1900),  ii.  151.  Gray, 
Brit.  Med.  Journ.  (1902),  i.  1121.  Leishnian,  ibid.  (1901),  i.  635  ;  ii. 
757.  Daniel,  ibid.  (1901),  i.  193.  Celli,  ibid.  (1901),  i.  1030.  Nuttall 
and  Shipley,  Journ.  of  Hyg.  i.  45,  269,  451  (with  literature).  Ross, 
Nature,  Ixi.  522;  "Mosquito  Brigades  and  how  to  organise  them," 
London,  1902.  Celli,  "  Malaria,"  trans,  by  Eyre,  London,  1900.  Lan- 
kester,  Brit.  Med.  Journ.  (1902),  i.  652.  Ewing,  Journ.  Exper.  Med.  v. 
429  ;  vi.  119.  Schaudinn,  Arbeit,  a.  d.  kaiserl.  Gesundheitsamtc,  xix  ; 
Arrjutinsky  Archiv  mikroskop.  Anat.  lix.  315  ;  Ixi.  331.  Ruge  in  Kolle 
and  Wassermann's  "  Handbuch  der  Pathogenen  Mikro-organism,"  Erganz- 
ungsband,  1907  (full  literature).  Ross,  Lancet  (1903),  i.  86.  Minchin, 
"The  Sporozoa,"  London,  1903.  Stephens,  art.  "  Blackwater  Fever," 
in  Allbutt's  "System  of  Medicine,"  vol.  ii.  pt.  ii.,  London,  1907. 
Laveran,  "Traite  du  paludisme,"  2nd  ed.,  Paris,  1907.  Stephens  and 
Christophers,  "  The  Practical  Study  of  Malarial  and  other  Blood  Para- 
sites," 3rd  ed.,  Liverpool,  1908.  Christophers  and  Bentley  (Blackwater 
Fever),  "  Scientific  Memoirs  published  by  the  Government  of  India," 
No.  35,  Simla,  1908. 

APPENDIX  D. — AMCEBIC  DYSENTERY. 

Lbsch,  Virchow's  Archiv,  Ixv.  196.  Cunningham,  Quart.  Journ.  Micr. 
•S'c.,  N.S.  xxi.  234.  Kartulis,  Virchow's  Archiv,  cv.  118  ;  Centralbl.  f. 
Bakteriol.  u.  Parasitenk.  ii.  745  ;  ix.  365.  Koch,  Arb.  a.  d.  k.  Gsndht- 
samte.  iii.  65.  Councilman  and  Lafleur,  John?  Hopkins  Hosp.  Rep.  (1891). 
ii.  395.  Maggiora,  Centralbl.  f.  Bakteriol.  u.  Parasitenk.  xi.  173.  Ogata, 
ibid.  xi.  264.  Schuberg,  ibid.  xiii.  598,  701.  Quincke  and  Roos,  Bed. 
klin.  Wchnschr.  (1893),  1089.  Kruse  and  Pasquale,  Ztschr.  f.  Hyg.  xvi. 
i.  Cieohanowsld  and  Nowak,  Centralbl.  f.  Bakteriol.  u.  Parasitrnk.  xxiii. 


BIBLIOGRAPHY  671 

1  !."•.  Howard  and  Hoover,  Am.  Joum.  Med.  Sc.  (1897),  cxiv.  150,  263. 
Harris,  'Vir<'1unrs  Archiv,  clxvi.  67.  Schaudinn,  Arbeit,  a.  d.  IcaiserL 
tlx.iilhlMtntt''.  (190:5),  xix.  r.-17.  Lesage,  Ann.  de  I'List.  Paste,,,-  (1905), 
xix.  9.  Kartulis  in  Kolle  aiul  Wasscrmaun's  "  Haudbuch  der  Pathogcnen 
Mikro-organismcn,"  Krgiin/ungsband,  1906  ;  Centralbl.  /.  Bakteriol. 
<>rij{).  (1904),  xxxvii.  527.  Musgrave  and  Clcgg,  "  Amccbas,  their 
Cultivation  and  Etiologic  Signification,"  Bureau  of  Government  Labora- 
tories, Manila,  1904  ;  Philip]).  Journ.  of  Science  (1906),  i.  Craig,  Jnvrn. 
////<-•/.  1  >;*•„*•*  (1908),  v.  324.  Viereck,  Bull.  dc.  Vlnst.  Pnxteur  (1907), 
v.'  819.  Hartmann,  ibid.  (1908)  vi.  100.  Werner,  Arch.  J  Set/.  ?/. 
7V"/"'/'/'//.'/.  xii.  11.  Noc,  Ann.  del'lnst.  Pasteur  (1908),  xxiii.  177. 

ArTKNDIX    E. — TllYPANOSOMIASlS,    KTC. 

C.KNKKAI,.  Lavcian  and  Mesnil,  "  Trypanosomes  et  trypanosomiasis," 
Paris,  Masson,  1904.  Minehin,  in  Clifford  Allbntt's  "System  of 
Medicine,"  2nd  cd.  vol.  ii.  pt.  ii.  p.  9,  London,  Macmillan,  1907. 
Schnudinn.  Arlxif.  <>.  <I.  hiiscrl.  Gcsundhcitsamte,  xx.  -387.  Mensc, 
"  Handbueh  der  Tropenkrankheiten,"  Leipzig,  1906,  Barth.  Novy  and 
Mai-NVal.  ,l»nr,,.  Inf.  Diseases,  ii.  '256.  Leishman,  Journ.  Hyg.  iv.  434. 
Minchin  and  Thomson,  Proc.  Roy.  Soc.  London,  B.  (1909),  Ixxxii.  273.  ' 
(Trypanosoma  Crnzi),  Chagas,  Ref.  in  Bull,  de  VInsl.  Pasteur  (1910), 
viii.  373. 

S  i .  i .  i •:  i- 1  N  < ;  Si<  K  N  KS  s.  — Mott,  Reports  of  the  Sleeping  Sickness  Commission 
"/  flic  Royal  Society,  pt.  vii.  No.  15,  London,  Bale,  Sons  &  Dannielsson, 
r.'Oi;.  Dutton  and  Todd,  Brit.  Med.  Journ.  (1903),  i.  304.  Duttou, 
.ind  Tod<l,  Tli<>,,ii>x(>n-Yates  Lab.  Rep.  v.  pt.  ii.  i.  ;  v.  pt.  ii.  97.  Button, 
Todd,  and  Christy,  ibid.  vi.  pt.  i.  p.  1.  Manson  and  Daniels,  ibid.  (1903), 
i.  1249.  Idem,  ibid.  (1903),  ii.  1461.  Low  and  Mott,  ibid.  (1904),  i. 
1000.  Bettenconrt,  Kopke,  Resende,  and  Mendes,  ibid.  (1903),  i.  908. 
Castellani,  Jfcjiorta  of  the  Sleejiimj  Sickness  Commission  of  the  Royal 
Surety,  No.  1,  i.  1,  London,  Harrison  &  Sons,  1903.  Bruce  and 
Nabarro,  ibid.  (1903),  No.  1,  ii.  11.  Bruce,  Nabarro,  and  Greig,  ibvl. 
(1903),  No.  4,  viii.  3.  Greig  and  Gray,  ibid.  (1905),  No.  6,  ii.  3. 
L'-ishman,  Journ.  Hyg.  iv.  434.  Minchin,  Gray,  and  Tulloch,  Reports  of 
thr  stc?itiiig  Sickness  Commission  of  the  Royal  Society,  No.  8,  xxi.  122, 
London,  H.M.  Stationery  Office,  1907.  Manson,  Brit.  Med.  Journ. 
(1903),  ii.  1249,  1461.  See  discussions  at  British  Medical  Association, 
/;/•/'.  M>'d.  Jonrn.  (1903),  ii.  637  ;  (1904),  ii.  365.  Thomas,  Thompson- 
y.ifc*  Lnb.  Urp.  vi.  pt.  ii.  1.  Kleine,  Deutsche  mcd.  Wchnschr.  (1909),  pp. 
I'.1.'.  924,  1257,  1956.  Bnice,  Hamerton,  Bateman  and  Mackie  (Sleeping 
Sirkncss  Commission  of  Royal  Society,  1908-9),  Proc.  Roy.  Soc.  London,  P.., 
Ixxxi.  40f>  ;  ibid.  Ixxxii.  pp.  5t»,  63,  256,  368,  480,  485,  498. 

LI.I>HM  \NIA  DONOVANI.— Leishman,  Brit.  Med.  Journ.  (1903),  i.  1252. 
/</•  in.  in  Clifford  ADbntt'a  " System  of  Medicine,"  2nd  ed.  vol.  ii.  pt.  ii.  226, 
London,  Macmillan,  1907.  Idem,  Mense,  "  Handbnch  der  Tropenkrank- 
heiten," iii.  591,  Leipzig,  Barth.,  1906.  Leishman  and  Statham,  Journ 
<>f  Roy.  Army  Med.  Corp*,  iv.  321.  Donovan,  Brit.  Med.  Journ.  (1903), 
ii.  79.  Rogers,  Quart.  Journ.  Micr.  Soc.  xlviii.  367.  Idem,  Brit.  J/"/. 
Jowrn.  (1904),  i.  1249  ;  ii.  645.  Idem,  Proc.  Roy.  Soc.  Ixxvii.  284. 
IVntley,  Brit.  Mcd.  Journ.  (1904),  ii.  653  ;  ibid.  (1905),  i.  705.  Chris- 
tophera,  Scientif.  Mem.  by  Off.  of  the  Mfd.  and  San.  Dept.  of  the  (,'ovt.  of 
Iii'lin,  Nos.  8,  11,  15.  Ross,  Brit.  Mcd.  Journ.  (1903),  ii.  1401.  See 
discussion  at  Brit.  Med.  Ass< ......  ///•//.  M«l.  Journ.  (1904),  ii.  642.  Patton, 


672  BIBLIOGRAPHY 

Sc,  Mem.  by  Officers  of  Med.  and  San.  Depts.  Gov.  India,  Calcutta,  1907, 
No.  27.  (Histoplasmosis),  Darling,  Journ.  Ex.  Med.  (1909),  xi.  515. 

LEISHMANIA  INFANTUM. — Nicolle,  Ann.  del'Inst.  Pasteur  (1909),  xxiii. 
361,  441.  See  also  references,  Bull,  de  Vlnst.  Pasteur,  viii.  164,  680. 
Pianese,  G-azz.  intern,  di  Medicin.  viii.  8. 

LEISHMANIA  TROPICA. — Wright,  J.  H.,  Journ.  Med.  Research,  x.  472. 
Marzinowsky,  Ztschr.  f.  Hyg.  Iviii.  327.  Row,  Quart.  Journ.  Med.  Sc. 
liii.  747.  Nicolle  and  Manceaux,  Ann.  de  Vlnst.  Pasteur,  xxiv.  673. 
Thomson  and  Balfour,  Journ.  Roy.  Army  Med.  Corps  (1910),  xiv.  1. 

PIROPLASMOSIS. — See  Minchin,  loc.  cit.  supra.  Koch,  Deutsche  med. 
Wchnschrft.  (1905),  No.  47  ;  Ztschrft.f.  Hyg.  u.  Infektionskrankh.  liv.  i. 
Nuttall,  Journ.  Hyg.  iv.  219.  Nuttall  and  Graham-Smith,  ibid.  v. 
237  ;  vi.  586. 

APPENDIX  F. — YELLOW  FEVER. 

Sternberg,  Rep'.  Am.  Pub.  Health  Ass.  xv.  170.  Sanarelli,  Ann,,  de 
Vlnst.  Pasteur,  xi.  433,  673,  753  ;  xii.  348.  Davidson,  art.  in  Clifford 
Allbutt's  "System  of  Medicine,"  vol.  ii.,  London,  1897.  Sternberur, 
Centralbl.  f.  Bakteriol.  u.  Parasitenk.  xxii.  145  ;  xxiii.  769.  Sanarelli, 
ibid.  xxii.  668.  Reed  and  Carroll,  Medical  News,  April  1899.  Reed, 
Journ.  of  Hyg.  ii.  101  (with  full  references).  Durham,  Thompson  -  Yates 
Laboratory  Rep.  (1902),  iv.  pt.  ii.  485.  Gorgas,  Lancet,  1902,  Sept.  9  ; 
1903,  March  28.  Marchoux,  Salimbeni,  and  Simond,  Ann.  de  Vlnst. 
Pasteur,  xvii.  665  ;  xx.  16,  104,  161.  Bandi,  Ztschr.  f.  Hyg.  (1904),  xivi. 
81.  Otto  and  Neumann,  Ztschr.  f.  Hyg.  (1905),  Ii.  Heft  3.  Reed,  Carroll, 
Agramonte,  Lazear,  Proc.  Amer.  Health  Ass.,  1900;  Journ.  Amer.  Med. 
Ass.,  Feb.  1901.  Carroll,  Neiv  York  Med.  Journ.,  Feb.  ]904  ;  Amer. 
Medicine  (1906),  xi.  383.  Thomas,  Brit.  Med.  Journ.  (1907),  i.  138. 


APPENDIX  G.— EPIDEMIC  POLIOMYELITIS. 

Landsteiner  and  Popper,  Ztschr.  f.  Immunitatsforsclmng  (Orig.)  (1902), 
ii.  377.  "Epidemic  Poliomyelitis,"  Report  on  New  York  Epidemic  of 
1907,  New  York,  1910.  Flexner  and  Lewis,  Journ.  Am.  Med.  A*s. 
(1909),  liii.  1639,  1913,  2095  (1910),  liv.  45,  1140,  1780.  Landsteiner 
and  Levaditi,  Comp.  rend.  Soc.  de  biol.  Ivii,  592,  787.  Levaditi  and 
Landsteiner,  ibid.  Iviii.  3,  11,  417.  Netter  and  Levaditi,  ibid.  Iviii. 
617,  855.  Levaditi,  Presse  med.  (1910),  43. 


APPENDIX  H. — PHLEROTOMUS  FEVEIJ. 


s 

"System  of  Medicine"  (1907),  ii.  (2)  345.  Ashburn  and  Craig, 
Philippine  Journ.  Sc.  Med.  ii.  93  (Ref.  in  Bull.  de.  Vlnst.  Pasteur 
(1907),  v.  773). 

APPENDIX  J. — TYPHUS  FEVER. 
Nicolle,  Ann.  de  Vlnst.  Pasteur  (1910),  xxiv.  243. 


INDEX. 


Al.rin,  196 

iinm unity  against,  520,  525 
Abscesses  (see  also  Suppuration) : 

bacteria  in,  202 

in  dysentery,  607 
A 1  isolate  alcohol,  fixing  by,  96 
Absorption  of  complement  test,  121 
Acid-fast  bacilli,  264,  278 

stain  for,  107 
A  i -ill     formation,     observation    of, 

51,  >2 
Ai-ijiiired  immunity  in  man,  528 

theories  of,  5-18 
Actinomyces,  16 

characters  of,  317,  318 

cultivation  of,  323 

inoculation  with,  327 

varieties  of,  325 
Actinomvooau,  317 

anaerobic  streptothricos  in,  326 

diagnosis  of,  327 

lesions  in,  321 

origin  of,  323 

Active  immunity,  514,  515 
Aerobes,  18 

culture  of.  ."7 

separation  of,  56 
JEstivo-autumnal  fevers.  .V.i:; 
African  tick  fever,  494 
Agar  media  (.sw  also  Culture  media), 

w 

separation  by,  60 
Agglutinable  substance,  543 
Agglutination  by  sera,  .'.  1 1 

in  relapsing  fever,  498 

methocuL  118 

of  b.  mallei,  313 

of  b.  typhosus,  etc.,  :',71 

43 


Agglutination    of    cholera    vibrio, 
458 

of  in.  melitensis,  492 

of  plague  bacillus,  487 

of  red  blood  corpuscles,  537,  542 

theories  regarding,  542 
Agglutinins,  measurement  of  group, 
120 

primary  (homologous),  375 

secondary  (heterologous),  375 
Agglutinogen,  543 
Agglutinoids,  543 
Aggressins,  189 
Air,  bacteria  in,  147 

examination  of,  for  bacteria,  147 
Albumose    of   anthrax,   immunity 

by,  343 
Albumoses,  193 

in  diphtheria,  408 
Alcohols,  higher,  fermentation  of, 

79 

Aleppo  boil,  636 
Alexines,  534,  557 
Amanita  phalloides,  toxin  of,  197 
Amboceptors,  536,  549 
Amcebic  dysentery,  602 
Amoibulae  of  malaria,  587 
Anaerobes,  18 

cultures  of,  65 

fusiform,  414 

separation  of,  63 

toxins  of,  60 

Anaerobic  Buchner  tubes,  66 
Anaerobic  Esmarch's  tubes,  64 
Anaerobic  fermentation  tubes,  65 
Anaerobic  plate  cultures,  liulloch'.s 

apparatus  for,  64 
Ana-fcthetio  leprosy,  299 


674 


INDEX 


Anaphylactin,  561 
Anaphylaxis,  192,  558 

mechanism  of,  561 

phenomena  in,  559 

reaction-bodies  in,  561,  562 

in  relation  to  rabies,  583 

supersensitiveness     to     tetanus, 
429 

toxic  phenomena,  192 

tubercular  sensitiveness,  287 
Aniline  oil,  dehydrating  by,  100 

water,  105 

Aniline  stains,  list  of,  101 
Animals,  autopsies  on,  145 

inoculation  of,  141 
Anthrax,  331 

anti-serum,  347 

bacillus,  332 
biology  of,  335 
cultivation  of,  333 
inoculation  with,  341 
toxins  of,  343 

diagnosis  of,  348 

in  animals,  337 

in  man,  341 

protective  inoculation,  346 

spread  of,  344 
Anti-abrin,  625 
Anti-anthrax  serum,  346 
Anti-bacterial  sera,  532 

properties  of,  533 
Anti-cholera  vaccination,  458 
Anti-diphtheritic  serum,  523 
Antiformin,  295 
Antigens,  521 
Antikorper,  513 
Anti-plague  inoculation,  486 
Anti-plague  sera,  486 
Antipneumococcic  serum,  239 
Antirabic  serum,  583 
Anti-ricin,  525 
Anti-sensibilisin,  561 
Antiseptics,  166 

actions  of,  168 

standardisation  of,  167 

testing  of,  166 
Antisera,    therapeutic    action    of, 

546 

Antistreptococcic  serum,  547 
Anti-substances,  specificity  of,  521 
Antitetanic  serum,  429 

preparation  of,  522  et  seq. 
Antitoxic  action,  nature  of,  526 
bodies  in  normal  tissues,  530 


Antitoxic  sera,  use  of,  525 
serum,  522 

standardisation  of,  524 
Antitoxins,     chemical     nature    of, 

526 

origin  of,  530 
Antitubercular  sera,  294 
Antityphoid  serum,  377 
Aortitis,  syphilitic,  506 
Appendicitis,  212 
Arthrospores,  question  of  occurrence 

of,  7 

Arthus  on  anaphylaxis,  559 
Artificial   immunity,    varieties   of, 

513  et  seq. 

Attenuation  of  virulence,  514 
Auer   and   Lewis   on   anaphylaxis, 

561 

Autoclave,  30 
Autolysis  of  bacteria,  188 
Autopsies  on  animals,  145 
Avian  tuberculosis,  276 

Bacilli,  acid-fast,  264,  278 
stain  for,  107 
anaerobic  fusiform,  444 
arthrosporous,  7 
characters  of,  14 
Bacillus  acidi  lactici,  21,  392 
aerogenes  capsulatus,  208,  442 
^Ertryk,  380,  384 
anthracis,  332 
botulinus,  438 
coli  anaerogenes,  393 
coli  communis,  lesions  caused  by, 
211  et  seq. 

agglutination  reactions,  354 

characters  of,  351 

culture  media  for,  51,  57,  154 

culture  reactions,  351  et  seq. 

gas  formation  by,  353 

isolation  of,  354 

morphological     characters    of, 
351 

pathogen icity  of,  356 

in  soil,  154,  155 

type  characters  of,  355 

in  water,  157,  161 
of  cholera,  447 
cloacae,  392 
of  Danysz,  384 
diphtheria,  397 
dysenterise  Shiga-Flexner,  385 
of  Emmerich,  393 


INDEX 


675 


Bacillus,  cntcritidis  (Gaertner),  383 
euteritidis  sporogenes,  389 

iu  soil,  154,  155 
of  Eschcrich,  350 
f.i-'.-alis  alcaligenes,  393 
'•!' glanders,  308 
of  hog  cholera,  380 
of  Hiippe,  393 
icteroides,  640 
<>f  influenza,  467 
Koch-Weeks,  219 
lactis  aerogenes,  208,  393 
lacunatus,  220 
of  leprosy,  299 
of  malignant  it'dema,  433 
Muller's,  219 
mycoides  in  soil,  153 
neapolitanus,  393 
o/.i-na-,  316 

oxytocus  perniciosus,  393 
paratyphosus,  382,  379 
of  plague,  475 
pneumonia',  226 
pseudo-diphtherieus,  410 
of  psittacosis,  384 
putrificens,  441 
I'V.ti-yaiHMi.s,  208 

agglutination  of,  541 

occurrence  of,  213 
pyogi'iu's  t'n-tidus,  202 
of  quarter-evil,  441 
of  rhinoscleroma,  315 
saccliam  !>utyricus,  441 
"f  sinegma,  380 
•  •Is,,  ft  sore,  257 
Mibtilis,  62 
suipestifer,  380 
of  syphilis,  50-'5 
tetaiii,  416 

of  Timothy  grass,  278 
of  tubercle, 
typhi  murium,  380 
of  typhoid,  35»; 
of  whooping-cough,  \l'i 
of  xerosis,  411 

Bacti-ria,  action  of  dead,  180 
aerobic  («<•<•  Ai-mln-.s),  18 
anaerobic  (scr  Anaerobes),  18 
In'ology  of,  17 
<  .i|»uluted,  3 
chemical  action  of,  22 

composition  of,  10 
classification  of,  11 
cultivation  of,  26 


-   Bacteria,  death  of,  166 

effects  of  light  on,  19 

food  supply  of,  17 

higher,  15 

lower,  12 

microscopic  examination  of,  91 

morphological  relations  of,  2 

motility  of,  8 

movements  of,  20 

multiplication  of,  4 

nitrifying,  24 

parasitic,  22 

pathogenic,  action  of,  175 
effects  of,  181 

saprophytic,  22 

separation  of,  56 

species  of,  24 

spore     formation     in     (so     U!M. 
Spores),  5,  62 

structure  of,  3 

sulphur-containing,  10 

temperature  of  growth  of,  19 

toxins  of,  187 

variability  among,  24 

virulence  of,  176,  517 
Bacterial  ferments,  23,  195 

pigments,  10 

protoplasm,  structure  of,  9 

treatment  of  sewage,  163 
Bactericidal  methods,  126 

powers  of  serum,  533 

substances,  534 
Bacteriological  diagnosis,  138 

examination  of  discharges,  136 
Bainbridge  on  agglutination,  121 
Beer  wort  agar,  52 
Beggiatoa,  16 
Behring  on  immunity,  429 
Besredka  on  anaphylaxis,  561 
Bile-salt  media,  50 
Bismarck-brown,  101 
Blackleg,  441 
Blackwater  fever,  599 
Blastophores  (malaria),  593 
Blood-agar  (see  also  Culture  media), 

43 
Blood,  examination  of,  72,  94 

in  malarial  fever,  584 

in  relapsing  fever,  494 

serum,  coagulated,  as  medium,  X) 
Blood-smeami  agar,   13 
Bone-marrow  in  leucocytosis,  182 
Bordet's  phenomenon,  534 
|  Bordet  and  Gengou's  medium,  44 


676 


INDEX 


Bordet  and  Gengou  on  whooping- 
cough,  472  et  seq. 

Botulism,  bacillus  of,  438 
toxin  of,  529 

Bouillon  (see  also  Culture  media),  33 

Bovine  tuberculosis,  274 

Bread  paste,  47 

Brieger  and  Boer,  193 

Brieger  and  Fraenkel,  187 

Buboes,  258 

Bubonic  pest,  475 

Buchner  on  alexines,  557 

Buchner's  anaerobic  tubes,  66 

Bulloch's  apparatus   for  anaerobic 
culture,  64 

Biitschli  on  bacterial  structure,  10 

Butter  bacilli,  acid-fast,  279 

Calmette,  486,  518,  525 
ophthalmo-reaction  of,  285 

Canary  fever,  646 

Canon  on  influenza,  467 

Cantani  on  influenza,  471 

Capaldi  and  Proskauer,  media  of, 
362 

Capsules,  staining  of,  109 

Carbol-fuchsin,  105 
-methylene-blue,  104 
-thionin-blue,  105 

Carbolic  acid  as  antiseptic,  173 

Carroll's  method  of  making  anae- 
robic cultures,  65 

Carter  on  relapsing  fever,  496 

Castellani  on  frambcesia,  511 

Cattle  plague,  569 

Cerebro-spinal    fluid,    examination 
by  lumbar  puncture,  73 

Chagas  on  trypanosomiasis,  629 

Chamberland   and  Roux,  attenua- 
tion of  b.  anthracis,  516 

Chamberland's  filter,  75 

Chemiotaxis,  20,  553 

Chitral  fever,  646 

Chlorine  as  antiseptic,  170 

Cholera,  446 
anti-sera,  458 
culture  methods,  44,  449 
immunity  against,  457 
inoculation  of  man  with,  457 
methods  of  diagnosis  of,  459 
preventive    inoculation    against, 

459 
-red  reaction,  451 

Cholera  carriers,  453 


Cholera  spirillum,  447 

distribution  of,  449 

inoculation  with,  453 

powers  of  resistance  of,  452 

relations  to  disease,  461 

toxins  of,  456 
Cladothrices  in  soil,  153 
Cladothrix,  16 

asteroides,  326 
Clubs  in  actinomyces,  320 
Coccaceae,  140 
Cocci,  characters  of.  12 
Coli-typhoid  bacteria,  391,  394 
Collodion  capsules,  preparation  of, 

144 

Colonies,  counting  of,  70 
Comma  bacillus,  446 
Commission  on  tuberculosis,  274 

on  vaccination,  527 
Complement,  534 

bacteriophilic,  538 

constitution  of,  534,  538 

deviation  of,  545 

method  of  estimating,  126,  130 

in  glanders,  313 

in  relation  to  precipitins,  546 

in  tuberculosis,  289 
Congestin,  559 
Conjunctivitis,  219 
Conradi-Drigalski  medium,  47 
i  Conradi's  picric  acid  method,  49 
i  Copeman  on  smallpox,  570 
Copper  sulphate  method,  109 
Cornet's  forceps,  94 
Corrosive  films  of  blood,  etc.,  95 
|  Corrosive  sublimate,  as  antiseptic, 
171 

fixing  by,  96 

Councilman  and  Lafleur  on  dysen- 
tery, 602 
Counting  of  colonies,  70 

dead  bacteria  in  a  culture,  133 

living  bacteria  in  a  culture,  71 
Cover-glasses,  cleaning  of,  94 
Cowpox,  relation  to  smallpox,  567 
Crescentic  bodies  in  malaria,  587 
Cultivation  of  anaerobes,  62 
Culture  media,  preparation  of,  31 
et  seq. 

agar,  37 

alkaline  blood  serum,  42 

blood  agar,  43 
serum,  40 

bouillon,  33 


INDK.X 


677 


( 'ulture  media,  preparation  of : 

1  >rra<l  paste,  47 

glucose  agar,  38 
broth,  36 
gelatin,  37 

•^Ivccrin  agar,  38 
broth,  36 

litmus  whey,  51 

LiifHer's  serum  medium,  41 

Marmorek's  serum  media,  42 

meat  extract,  32 

milk,  46 

peptone  gelatin,  36 
solution,  39 

potatoes,  45 

serum  agar,  43 
Cultures,  destruction  of,  89 

filtration  of,  74 

from  organs,  137,  145 

hanging-drop,  aerobic,  69 

incubation  of,  85 

microscopic  examination  of,  91 

permanent  preservation  of,  88 

plate,  59 

pure,  54 

"shake,"  81 

Cutaneous  tuberculin  reaction,  285 
<  'utting  of  sections,  97 
( 'ystitis,  212,  255 
Cytases,  553 
(  'ytoly  tic  sera,  538 

1 )  ni\  >/.'s  bacillus,  384 

I  )arling  on  hisLoplusma  capsulatum, 

637 

Dead  cultures,  counting  of,  133 
I)e  Bary,  definition  of  species,  24 
Decolorising  agents,  103 
Deep  cultures,  65 
Dehydration  of  sections,  100 
Delepine,  agglutination  method,  119 
Delhi  sore,  636 
Deneke's  spirillum,  465 
Dengue  fever,  646 
Deviation  of  complement,  126,  130, 

546 

Dextrose-free  bouillon,  80 
Diagnosis,  bacteriological,  135,  138 
Dieudonne's  medium,  44 
Diphtheria,  396 

diagnosis  of,  412 

immunity  against,  523 

origin  and  spread  of,  398 

paralysis  in,  397,  405 


Diphtheria,   results  of  treatment, 

546 
Diphtheria  bacillus,  action  of,  402 

bacilli  allied  to,  409 

characters  of,  397 

distribution  of,  398 

fermentation  reactions  of,  402 

inoculation  with,  404 

isolation  of,  412 

powers  of  resistance  of,  403 

staining  of,  115,  403 

toxins  of,  191,  405,  407,  409 

variations  in  virulence  of,  408 
Diphtheroid  bacillus,  410 
Diplo-bacillus  of  conjunctivitis,  220 
Diplococcus,  12 

catarrhalis,  247 

crassus,  247 

endocarditidis  encapsulate,  216 

intracellularis  meningitidis,  242 

mucosus,  247 

pharyngis,  247 

pneumonias,  227 
Disaccharides,  79 
Disturbances     of    metabolism     by 

bacteria,  185 

Doerr  on  phlebotomus  fever,  646 
Dorset's  egg  media,  267 
Dreyer  and  Jex-Blake  on  aggluti- 
nation, 543 

Drigalski-Conradi  medium,  47 
Drying  of  sera,  etc. ,  in  vacuo,  84 
Ducrey's  bacillus,  257 

cultivation  of,  258 
Dum-Dum  fever,  630 
Durham's  fermentation  tubes,  81 
Dysentery,  amoebic,  602 

bacteria  in,  384 

characters  of  amoeba  of,  602 
cultivation  of,  605 
distribution  of,  606 
inoculation  experiments,  607 
Dysentery,  methods  of  examination 
in,  385 

East  coast  fever  in  cattle,  638 

Eberth's  bacillus,  350 

Eel  serum,  198 

Egg  media  for  tuberculosis,  267 

Ehrlich  on  ricin  and  abrin,  520,  525 

on  toxins,  198 

rosindol  reaction,  83 

side-chain    theory    of   antitoxin 
formation,  649 


678 


INDEX 


Eisenberg  on  anthrax,  190 
Eisner's  medium,  51 
Embedding  in  paraffin,  97 
Emmerich's  bacillus,  393 
Empycnia,  234,  470 
Endows  medium,  49 
—-Endocarditis,  bacteria  in,  216 

Endotoxins,  522.    See  Intracellular 

toxins. 

Enhaeinospores  (malaria),  587 
Entamoeba  coli,  603 
Entamceba  histolytica,  603 

cultivation  of,  605 
Enteritis,  dysenteric,  386,  606 
Epidemic  cerebro-spinal  meningitis, 
242 

poliomyelitis,  644 
Eppinger's  streptothrix,  326 
Ermengem  on  botulism,  438 

stain  for  flagella,  110 
Erysipelas,  218 
Escherich's  bacillus,  350 
Esmarcli's  roll-tubes,  60 

anaerobic,  63 

Exaltation  of  virulence,  517 
Examination  of  water,  156 
Exhaust-pump,  75 
Exotospores  (malaria),  586 
Extracellular  toxins,  190,  522 

False  membrane,  212,  397 

Farcy,  307 

Fawcus'  picric  acid  method,  49 

Feeding,  immunity  by,  520 

Fermentation  by  pneumo-bacillus, 

232 

by  bacillus  coli,  352,  392 
by  b.  diphtherias,  402    - 
methods  of  observing,  78 
of  sugars  by  bacteria,  79 
test  of  bacterial  action,  79 
tubes,  81 
anaerobic,  65 

Ferments  formed   by  bacteria,  23, 

195 
in  diphtheria,  402,  408 

Ferrata  on  complement,  534 

Fever,  185 

Film  preparations,  dry,  93 
wet,  95 
staining  of,  102 

Filter,  porcelain,  gelatined,  193 

Filtration  of  cultures,  74 

Finkler  and  Prior's  spirillum,  464 


Fish,  tuberculosis  in,  277 

Fixateurs,  553 

Fixation  of  complement,  130 

of  tissues,  96 
Klagella,  nature  of,  8 

staining  of,  110 
j   Flagellated  organisms   in   malaria, 

592 
Flexner  on  epidemic  poliomyelitis, 

644 

Fliigge,  15 

Food-poisoning  bacilli,  379 
Forceps  for  cover-glasses,  94 
Ford    Robertson     on     diphtheroid 

bacilli,  410 

Formalin  as  antiseptic,  171 
Forster  on  typhoid  fever,  368 
Foth's  dry  inallein,  314 
Fraenkel's  pneumococcus,  226,  229, 
230 

stain  for  tubercle,  107 

on  whooping-cough,  474 
Framboesia,  spirochpetes  in,  511 
Frankland  on  water  bacteria,  160 
Fraser,  T.  R.,  518,  525,  532 
Friedberger  on  anaphylaxis,  562 
Friedliinders  pneumobacillus,  226, 

232 

Frisch  on  rhinoscleroma,  316 
Frothingham  on  Negri  bodies,  577 
Fuchsin,  carbol-,  105,  108 
Fusiform  anaerobic  bacilli,  444 

Gallstones    in  relation   to  typhoid 

fever,  364 

|  Gamale'ia  on  pneumonia,  235 
i  Gametocytes  (malaria),  587 
Gangrenous  emphysema,  433,  437 

pneumonia,  470 
Gas  formation,  observation  of,  50 

82 

Gas-regulator,  86 

Gay  and  Adler  on  anaphylaxis,  561 
|   Geissler's  exhaust-pump,  75 
Gelatin  media,  36 

separation  by,  56 
Gelatined  porcelain  filter,  193 
General       paralysis,      diphtheroid 

bacilli  in,"  410 

Wasserman  reaction  in,  510 
j  Gentian-violet,  105 
I  Germicides,  166 
i  Geryk  pump,  85 
i  Giemsa's  stain,  115 


INDEX 


G79 


Giemsa's  stain  for  spirochrotes   in 

films,  115 
Glanders,  306 

diagnosis  of,  314 

in  horses,  307 

in  man,  307 

loii ins  in,  312 
Glanders  bacillus,  308 

agglutination  of,  313 

inoculation  with,  311 
Globulin,  constituent  in  antitoxin, 

532 
Glossina  morsitans,  618 

palpalis,  624 
( llucosc  media,  3»>  r.t  .*••"/. 
Clucosides,  fermentation  of,  79 
Glycerin  media,  36  et  scq. 

potato  as  culture  medium,  46 
(Jolgi  on  malaria,  585 
Gonidia,  16 
Gonococcus,  characters  of,  249 

comparison  with  meningococcus, 
252 

culture  methods,  42,  43 

inoculation  with,  253 

toxin  of,  253 
Gonorrhoea,  249 
Gonorrhoeal  conjunctivitis,  255 

endocarditis,  256 

septicaemia,  256 
Craham-Smith  on  identification  of 

bacilli,  4 
Gram's  method,  105 

Kiihne's  modification  of,  106 

Much's  modification  of,  265 

Wcigrrt's  modification  of,  106 
(irassberger    and   Schattenfroh   on 
quarter-evil,  441,  442 

on  symptomatic  anthrax,  192 
Grease,  566 

Civrnticld  on  anthrax,  343,480,516 
Group  agglutinins,  measurement  of, 

120 
Griiber  and  Durham's  phenomenon, 

541 

Guarnieri  bodies  in  smallpox,  570 
Gulland  (methods),  95,  99 

Hsemamceba  Danilewski,  594 

malaria;,  594 

pr;«'cpx,  594 

relicta,  594 

vivax,  594 
Hrematozoon  malaria? ,  585 


Hremolytic  sera,  536 

Hremolytic  tests,  methods  of,  128, 

538 

Ha  triune  on    anti-cholera  inocula- 
tion, 459 
Haffkine's      inoculation      method 

against  plague.  486 
Haltcridium,  592,  594 
Hanging-drop  cultures,  69 

examination  of,  91 
Hankin,  344 

Hansen,  leprosy  bacilli,  299 
Harrison's    method     for    counting 

bacteria,  134 
Hesse's  tube,  148 
Hiss's  serum  water  media,  47 

method  of  capsule  staining,  109 
Histoplasma  capsulatum,  637 
Hermann's  bacillus,  410 
Hog  cholera,  380 

Hogyes    on    treatment    of   hydro- 
phobia, 582 
Horsepox,  569 

Houston  on  bacteriology  of  soil,  152 
Hiippe,  7,  15 
Hiippe's  bacillus,  393 
Hydrogen,  supply  of,  62 
Hydrophobia,  573 

diagnosis  of,  583 

Negri  bodies  in,  576 

prophylactic  treatment  of,  575, 
579 

virus  of,  579 
Hypodermic  syringes,  143 

Immune-bodies,  534 

origin  of,  536 

Immunity  (sec  also  Special  Diseases), 
512 

acquired,  theories  of,  548 

active,  515,  516 

artificial,  513 

by  feeding,  520 

by  toxins,  518 

methods,  515 

natural,  555 

passive,  514,  520 

unit  of,  524 

Impression  preparations,  138 
Incubators,  85 
Indol,  formation  of,  82 
Infection,     conditions    modifying, 
175 

nature  of,  179 


G80 


INDEX 


Inflammatory    conditions    due    to 

bacteria,  183 
Influenza,  467 

lesions  in,  471 

sputum  in,  469 
Influenza  bacillus,  467 

cultivation  of,  43,  468 

inoculation  with,  471 

pseudo-bacilli,  470 
Inoculation,  methods  of,  141 

of  animals,  141 

of  tubes,  55 

protective,  518  etseq. 

separation  by,  61 
Intestinal  changes  in  cholera,  449 

amoebic  dysentery,  606 

bacterial  dysentery,  387 

typhoid  fever,  365 
Intestinal  infection  in  cholera  (ex- 
perimental), 453 
Intracellular  toxins,  188,  522 
Involution  forms  in  bacteria,  5 
Iodine  solution,  Gram's,  106 

terchloride,  523 

as  antiseptic,  170 
lodoform  as  antiseptic,  173 
Issaeff,  520 
Ivanoff's  vibrio,  462 

Japanese  dysentery,  389 
Jenner  on  vaccination,  565 
Jenner's  stain,  113 
Johne's  bacillus,  279 
Joints,  gonococci  in,  255 

Kala-azar,  630,  635 
Keratitis,  syphilitic,  509 
Kipp's  apparatus,  63 
Kitasato   on   bacillus  of  influenza, 
467 

of  plague,  475 

of  tetanus,  416  etseq. 
Klebs-Lbffler  bacillus,  396 
Klein,  378,  570 
Klemperer  on  pneumonia,  239 
Klimenko  on  whooping-cough,  474 
Knapp  and  Novy  on  relapsing  fever, 

498 
Koch  on  avian  tuberculosis,  276 

bacillus    of    malignant    redema, 
433 

bovine  tuberculosis,  274 

cholera  spirillum,  446 

cultivation  of  b.  anthracis,  333 


Koch  on  tubercle  bacillus,  260 
Koch's  blood  serum,  40 

glass  plates.  59 

leveller  for  plates,  59 

new  tuberculin,  288 

tuberculin,  284 

"tuberculin  0,"  and  "R,"  288 
Koch- Weeks  bacillus,  219 
Korn's  acid -fast  bacillus,  279 
Kraus  on  cholera,  461 
Kruse  and  Pasquale  on  dysentery, 

607 
Kubel-Tiemann     litmus     solution, 

48 
Kiihne's  methylene-blue,  105 

modification  of  Gram's  method, 
106 

Lactose  fermenters,  391 
Lamb  on  relapsing  fever,  498 
Laveran  on  malarial  parasite,  585 
Leishman-Donovan  bodies,  630 

cultivation  of,  633 
Leishman's  opsonic  technique,  121 

serum   method  for  staining  try- 
panosomes,  611 

stain,  114 

Leishman  on  tick  fever,  502 
Leishmania  donovani,  630 

infantum,  635 

tropica,  635 
Leishmaniosis,  630 
Lenses,  91 
Lepra  cells,  299 
Leprosy,  297 

bacillus,  299 

distribution  of,  301 
staining,  107,  300 

diagnosis  of,  304 

etiology  of,  302 

Leprosy-like  disease  in  rats,  30-°> 
Leptothrix,  16 

Lesions  produced  by  bacteria,  181 
Leucocytosis,  182,  552 
Leucomaines,  187 

Levaditi's    collodion    sac    method, 
503 

method  for  staining  spirocheetes, 
112 

on  tick  fever,  503 

and    Mclntosh    on   Sp.    pallida, 

507 

Levy  on  streptococci,  207 
Litmus  media,  39 


INDEX 


081 


Litmus  solution,  Kubel-Tiemann's, 
48 

\vlu-y,  51 

Liver  aliMv<s  in  dvH-ntcry,  60 
Lockjaw,  415 
l/>tll.Vs  bacillus,  3!)G 

methylene-blue,  104 

scrum  medium,  41 

and    Schut/c'    glanders    bacillus, 

306 

Lbsch,  amoeba  of,  602 
Lumbar  puncture,  73 
Lustgarten's  bacillus,  503 
Lustig's  anti-plague  serum,  487 
Lymph,  vaccine,  568 
Lymphangitis,  212 
Ly.sa-mia  in  l)lackwater  fever,  599 
Lysogenic  action  of  serum,  534 

towards  blood  corpuscles,  536 

MacConkey's  bile-salt  media,  50 
medium,    use    of   in    dysentery, 

386 

on  coli-typhoid  group,  391 
in  examining  water,  157 
in  paratyphoid  fever,  381 
MacDonald  on  meningitis,  244 
MeFadyean  on  glanders,  313 

methylene-blue  reaction  in  anth- 

rax>  333 

Macrocytase,  553 
Macrophages,  552 
Madura  disease,  328 
Malaria,  cycle  in  man,  586 

in  mosquito,  592 
pathology  of,  597 
prevention  of,  596 
question    of   immunity    against, 

598 
Malarial     fever,     examination     of 

blnod  in,  600 
malignant,  587,  595 
mosquitoes  in,  596 
parasite,  585 
inoculation  of,  586 
.staining  of,  Irishman's  method, 

114 

Romanowsky  methods,  113 
varieties  of,  593 

Malignant  oedema,  bacillus  of,  433 
diagnosis  of,  438 
immunity  against,  438 
Malignant  pustule,  342 
Mallein,  314 


Malta  fever,  488 

methods  of  diagnosis,  493 
spread  of  disease,  4(.*i 

Mann's  method  of  fixing  sections,  99 
Manson,  584 

Manteufel  on  relapsing  fever,  49'J 
Maragliano's  anti-tubercular  serum, 

294 
Marchiafava  and   Celli  on  malaria, 

584 
Marmorek  on  streptococci,  210 

antistreptococcic  serum,  533 
Marmorek's  serum  media,  42 

antitubercular  serum,  294 
Martin,  C.  J.,  on  toxins,  193 

on  antitoxins,  532 
Martin,     Sidney,     on     alburn^,  s. 
etc.,  194 

on  anthrax,  343 

on  diphtheria,  408 
Massowah  vibrio,  462 
Measuring  bacteria,  140 
Meat  extract,  32 

Meat-poisoning   by   bacillus    botu- 
linus,  438 

by  Gaertner's  bacillus,  383 
Mediterranean  fever,  488 
Meningitis,  bacteria  in,  247 

epidemic  cerebro- spinal,  202,  242 

in  influenza,  470 

pneumococci  in,  234 

posterior  basal,  245 
Meningococcus,  242 

allied  diplococci,  247 

anti-sera,  246 

comparison  with  gonococcus,  252 

serum  reaction,  245 
Mercury  perchloride  as  antiseptic, 

171 

Merozoites  in  malaria,  587 
Metabolism,    disturbances    of,     by 

bacteria,  185 

Metachromatic  granules,  9 
Metacoccaceae,  141 
Metchnikoff  on  cholera  in  rabbits, 
454 

relapsing  fever,  454 

on  syphilis,  508 

MetchnikofTs  phagocytosis  theory, 
552 

spirillum,  464 
Methylene-blue,  104,  105 

reaction  in  anthrax,  McFadvean, 
333 


682 


INDEX 


Methyl-violet,  101 

Meyer    and     Ransom    on    tetanus 

toxin,  427 

Micrococci  of  suppuration,  202 
Micrococcus,  12 

of  gonorrhoea,  249 

melitensis,  489 

pyogenes  tennis,  202 

tetragenus,  209 

lesions  caused  by,  213 

urese,  21 

Microcytase,  553 
Microphages,  552 
Microscope,  use  of,  90 
Microtomes,  97 
Migula,  15 

Mikulicx,  cells  of,  315 
Milk  as  culture  medium,  46 
Minchin        on       trypanosomiasis, 

613 
Mceller's    Timothy-grass    bacillus, 

278 

Mb'ller's  stain,  for  spores,  109 
Monosaccharides,  79 
Moore's    medium    for  coli-typhoid 

bacilli,  51 

Morax,  bacillus  of,  220 
Mordants,  103 

Morgan's  bacillus  No.  1,  390 
Mosquitoes,  in  malaria,  592,  596 

role  in  yellow  fever,  641 
Moulds,  media  for  growing,  52 
Much's     modification     of    Gram's 

method,  265 
Muencke's  filter,  77 
Miiller's  bacillus,  219 
Musgrave  and  Clegg  on    amoebic 

dysentery,  605,  606,  608 
Mycetoma,  328' 
Myelocytes,  neutrophile,  181 

Nagana,  618 

Nasgar  medium,  43 

Natural  immunity,  555 

Neelsen's  stain  for  tubercle,  108 

Negative   phase  in   immunisation, 
291,  519 

Negri  bodies  in  rabies,  576 

Neisser    and  Wechsberg's  bacteri- 
cidal method,  127 

Neisser's  gonococcus,  249 
stain  for  b.  diphtheria,  115 

Nencki,  11 

Neuroryctes  hydrophobice,  578 


Neutral-red  as  indicator  for  media, 

50 
use  of,  39 

Avith  b.  coli,  353 
Neutrophile  leucocytes,  181 

myelocytes,  181 
Nicolaier,  tetanus  bacillus,  415 
Nicolle   on    Leishmania  infantum, 

635 

on  Leishmania  tropica,  636 
on  typhus  fever,  648 
Nicolle's    modification    of   Gram's 

method,  107 

Nikati  and  Rietsch  on  cholera,  453 
Nitrifying  bacteria,  24 
Nitroso-indol  body,  82 
Nordhafen  vibrio,  464 
Novy  on  relapsing  fever,  495,  498 
Novy  and  MacNeal,  medium  for  cul- 
ture of  trypanosomes,  45,  612 

Obermeier's  spirillum,  494 
(Edema,  malignant,  433 
Ogata's  dysentery  bacillus,  389 
Ogston,  202 
Oil,  aniline,  for  dehydrating,  etc., 

100 

Oil  immersion  lens,  91 
Ookinete,  592 
Ophthalmic     tuberculin     reaction, 

285 
Opsonic  action,  nature  of,  539 

technique,  121 
Opsonins,  122 

absorption  of,  540 

in  tuberculosis,  289 

therrnolabile,  540 

thermostable,  540 
Organisms  lower  than  bacteria,  2, 

640 

Oriental  plague,  475 
Osteomyelitis,  217 
Otitis,  234,  470 

Oxygen,  nascent,  as  antiseptic,  170 
Ozoena  bacillus,  316 

Pappataci  fever,  646 
Parabolic  condenser,  504 
Paracoccaceae,  141 
Para-colon  bacillus,  382 
Paraffin  embedding,  97 
Paratyphoid  bacillus,  379,  382 
Park  and  Collins  on  agglutination, 
-121 


INDEX 


683 


Park  ami  Williams   on  diphtheria 
tuxin,  407 

.  .">]»; 

Passive  immunity.  ."•]  1,  520 
Pasteur  on  exaltation  of  virulence 
of  liaeteria,  517 

on  hydrophobia,  575 

nil  vaccination   against  anthrax, 
846 

septio'inic  «lr,  433 
Pathogenicity  of  bacteria,  175 
Peptone  gelatin  (sec  Culture  media), 
36 

solution,  39,  451 

Periostitis,  acute  suppurative,  217 
Peritonitis,  212,  255 
iVrlsucht,  261 
Pesti^  major,  481 

minor,  481 
Pctri's  arid-last  liacillus,  279 

capsules,  57 

sand-filter  for  examining  air,  149 
Petruschky's  litmus  whey,  52 
Pettenkofer  on  cholera,  455,  462 

rtcfier,  20 

ITciH'er  on  anti-serum,  534 

cholera,  457 

influenxa,  466. 

tvphoid,  369 
Pfeiffer'a  media,  l:; 
Pl'ciifer's    phenomenon,     457,    533, 

534 

Phagocytes.  ls-j 
Phagocytosis  theory  of  Metchnikoll', 

552 

Phenol  broth,  154 
Phenol-phthalein  as  indicator,  34 
Phenomenon  of  Bordet,  534 

Crul •.«•!•  and  Durham,  541,  542 

I'fcill.-r,  457,  533,  534 
Phlebotomus  fever,  646 
Picric  acid  media,  49 
Pigment.-,  bacterial,  10 
Pipettes,  71,  116,  119,  124 
Piroplasmata  as  causes  of  disease, 

638 

I'iroplasmosis,  637 
Pitfield's  flagella  stain,  110 
1' la^uc.  bui-illus  of,  475  tt  seq. 

Hafl'kinc's     inoculation    against, 
486 

immunity  against,  485 

infn-tioii  in,  482 

involution  forms,  478 


Plague,  part  played  by  rat  fleas  in 
the  spread  of,  484 

preventive    inoculation    again>t. 
486 

serum  diagnosis,  187 

stalactite  growths  of,  478 

varieties  of,  481 
Plasmolysis,  9 
Plate  cultures,  agar,  60 

gelatin,  56 
Platinum  needles,  54 
Pneumobacillus(Friedliinder's),227, 

232  ct  ,sv  Y. 

Pneumococcus     (Fraenkel's),     227, 
229  ft  seq. 

capsulation  of,  230 

culture  methods,  43 

fermentation  reactions  of,  231 

immunity  against,  239 

in  endocarditis,  216 

lesions  caused  by.  233 

relation  to  streptococci,  231 

toxins  of,  238 
Pneumonia,  bacteria  in,  225 

gangrenous,  470 

in  influenza,  469 

methods  of  examination  of,  241 

septic,  225 

varieties  of,  224 
Polar  granules,  8 
I  Poliomyelitis,  644 
Polysaccharides,  79 
Positive    phase    in    immunisation, 

292,  519 

Potassium   permanganate  as  anti- 
septic, 173 

Potatoes  as  culture  material,  45 
Poynton  and  Payne  on  acute  rheu- 
matism, 220 
Precipitinogen,  545 
Precipitins,  544 
Precipitoid,  546 
Preparations,  impression,  138 
Protective  inoculation,  578  ctscq. 
Proteosoma,  594 

Protozoa  described  in  hydrophobia, 
578 

smallpox,  570 
Protozoon  malaria,  585 
Prowazek  on  smallpox,  571 

on  the  trypanosomata,  613 
Pseudo-diphtheria  bacillus,  410 

-tuberculosis  streptothrices,  327 
Psittacosis  bacillus,  384 


684 


INDEX 


Ptomaine  poisoning,  379 
Ptomaines,  187 
Puerperal  septicaemia,  212 
Pus,  examination  of,  94,  222 
Pustule,  malignant,  342 
Pyaemia,   212  ct  seq. 

"nature  of,  201 

Pyogenic  cocci,  culture  of.  133 
Pyrogallate  of  potassium  for  anae- 
robic cultures,  63 
Pyrogallol  saturated  tubes,  68 

Quartan  fever,  594 
Quarter-evil,  bacillus  of,  441 
Quotidian  fever,  593 

Rabies,  573 

Rabinowitch's     acid-fast     bacillus, 

279 

Rat  viruses,  384 
Rauschbrand  bacillus,  441 
Ray-fungus  (actinomyces),  317 
Reaction  of  media,  standardising  of, 

33 

Receptors,  549 
Recovery  from  disease,  513 
Red  stains,  101 
Red-water  fever  in  cattle,  638 
Reichert's  gas-regulator,  86 
Relapsing    fever,    agglutination   of 

spirillum,  498 
i  bactericidal  serum  in,  498 

spirillum  of,  etc.,  495 

transmission  of,  499 

varieties  of,  498 

Reversibility  of  toxin-antitoxin  re- 
action, 528 

Rheumatism,  acute,  220 
Rhinoscleroma,  bacillus  of,  315 
Richet  on  anaphylaxis,  559 
Ricin,  196 

immunity  against,  520,  525 
Rivers,  bacteria  in,  160 
Robin,  196 
Rock  fever,  488 
Roll-tubes,  Esmarch's,  60,  63 
Romanowsky  stains,  113 
Rosenbach    (bacteria    in    suppura- 
tion), 202 

Rosindol  reaction  (Ehrlich),  83 
Ross  on  malaria,  585 

thick  film   method   for  malarial 

parasite,  600 
Roux  on  antitoxic  sera,  523,  525 


Roux  on  syphilis,  508 

and  Yersin  (diphtheria),  407    ct 
scq. 

Sabouraud's  media,  52 

Safranin,  101 

Salt-agar  as  medium  for  b.  pcstis 

476 

Sanarelli  (typhoid  fever),  356 
Sanderson,  Burdon,  516,  569 
Saprophytes,  175 
Sarcina,  14 
Sausage   poisoning,    bacillus   botu- 

linus  in,  438 

Schaudinn  on  biology  of  trypano- 
somes,  614 

on   amoebae   of  dysentery,    602, 
604,  605 

on  morphology  of  spirilla,  616 

on  spirochaete  pallida,  503 

on  spirillum  Ziemanni,  616 
Schizogony,  586 
Schizomycetes,  3 
Schizonts,  588 
Schizophyceae,  3 
Schizophyta,  3 
Schiitfner's  dots,  114 
Sclavo's  anti-anthrax  serum,  347 
Scorpion  poison,  197 
Section-cutting,  97 
Sections,  dehydration  of,  100 
Sedimentation  methods,  118 

test  for  typhoid,  371 
Seitenketten,  549 
Sensibilisinogen,  561 
Septicaemia,  nature  of,  201 

puerperal,  212 

sputum,  225 

Septicemie  de  Pasteur,  433 
Septic  pneumonia,  225 
Sera,  hgemolytie,  536 
Serum  agar,  43 
Serum,  agglutinative  action  of,  541 

anaphylaxis,  558 

antibacterial,  532 

anti-cholera,  458 

antidiphtheritic,  523 

anti-plague,  487 

antipneumococcic,  239 

antirabic,  583 

antistreptococcic,  533 

antitetanic,  429 

antitoxic,    preparation    of,     523 


INDEX 


685 


Serum,  antitubercular,  294 

antityphoid,  377 

bactericidal  action  of,  533 

blood  (see  Culture  media),  40 

diagnosis,  541 
methods,  118 
of  syphilis,  131,  510 
of  typhoid,  371 

inspissator,  41 

Ivsogenic  action  of,  534 

towards  blood  corpuscles,  536 
Scrum  disease,  563 
Serum  media,  40 
Serum-water  media,  47 
Seven-day  fever,  646 
Sewage,  bacterial  treatment  of,  163 

contamination  of  water  by,  160 
Shake  cultures,  81 
Shanghai  fever,  646 
Sheep-pox,  569 
Shiga's  bacillus,  384 
Side-chain  theory,  Ehrlich's,  549 
Sleeping  sickness,  621 
Slides  for  hanging-drops,  69 
Sloped  cultures,  aerobic,  53 

anaerobic,  68 
Smallpox,  564 

bacteria  in,  569 

Guarnieri  bodies  in,  570 
Smegma  bacillus,  280 
Smith's,  Lorrain,    serum    medium, 

42 
Smith,  Theobald,  phenomenon  of, 

559 
Snake  poisons,  197 

activating  of,  by  serum,  197 

constituents  of,  197 

immunity  against,  518 
Sobernheim's   anti-anthrax    serum, 

347 
Soft  sore,  257 

bacillus  of,  257 

culture  methods,  43,  258 
Soil,  examination  of,  for  bacteria, 

151 
Soudakewitch   on    relapsing   fever, 

498 

Spirilla,    characters    of    (xee    also 
Vibrio),  14,  616 

like  cholera  spirillum,  462 
Sjiirillosis  in  animals,  4'.C> 
Spirillum  Metchnikovi,  464 

of  cholera,  447 

Deneke,  465 


Spirillum  Duttoni,  502 

Finkler  and  Prior,  464 

Miller,  465 

Obermeieri,  494,  503 

relapsing  fever,  inoculation  with, 

etc.,  496 
Spirochsete,  15,  503,  616 

gallinarum,  502 

pallida,  507 

staining  of,  112,  115 

pallidula,  511 

pertenuis,  511 

refringens,  504 
Spirochsetes,  diseases  due  to,  494 

in  syphilis,  503 

in  tick  fever,  503 

in  yaws,  511 

staining  of,  in  films,  115 

staining  of,  in  sections,  112 
Spironema  pallidum,  503 
Splenic  fever,  331 
Spore  formation,  arthrosporous,  7 

endogenous,  5 

in  b.  anthracis,  336 
Spores,  staining  of,  109 
Sporoblasts,  593 
Sporocyst  (malaria),  593 
Sporogony  (malaria),  593 
Sporozoites,  586.     See  Schizonts 
Sporulation   of   malarial    parasite, 

586 
;  Sputum,  amoebae  in,  607 

influenza,  469,  472 

in  plague,  481 

in  pneumonia,  228 

phthisical,  268,  281,  295 

septicaemia,  225 
Staining  methods,  101  et  seq. 

of  capsules,  Hiss's  method,  109 
Welch's  method,  109 
Richard  Muir's  method,  110 

of  flagella,  110 

of  leprosy  bacilli,  300 

of  spores,  109 

of  tubercle  bacilli,  107 

principles,  101 
Stains,  basic  aniline,  101 
Standard  of  immunity,  ">2 1 
Standardising  reaction    of  media, 

33 
Staphylococci,    lesions   caused    by 

212 
Staphylococcns,  12 

cereus  albus,  204 


686 


INDEX 


Staphylococcus,  cereus  flavus,  204 

pyogenes  albus,  204 

aureus,  characters  of,  202 

inoculation  with,  210 
citreus,  202 

Steam  steriliser,  Koch's,  28 
Stegomyia  f'asciata,  641 
Sterilisation  by  heat,  27  et  *cq. 

at  low  temperatures,  30 

by  steam  at  high  pressure,  29 
Streptococci  in  diphtheria,  400 

in  false  membrane,  212 

hsemolytic  action  of,  207 

lesions  caused  by,  212 

varieties  of,  206 
Streptococcus,  12 

anginosus,  207 

brevis,  206 

conglomerates,  206,  207 

equinus,  207 

erysipelatis,  218 

fsecalis,  159,  207 

longus,  206 

mitior,  207 

mucosus,  231 

pneumonia,  226 

pyogenes,  characters  of,  204 
inoculation  with,  222 
in  air,  151 
in  soil,  154 

salivarius,  206 

saprophyticus,  207 
Streptothrices     allied      to    actino- 

myces,    326 
Streptothrix,  16 

actinomyces,  318 

Streptothrix,  anaerobic,  in  actino- 
mycosis,  325 

madune,  329 
Subcultures,  54 
Sugars,  classification  of,  79 

fermentation  of,  78 

by  b.  coli  group,  392 
Sulphurous  acid  as  antiseptic,  172 
Summer  diarrhoea,  bacteria  in,  390 
Supersensitiveness,  558.     See  Ana- 

phylaxis 
Suppuration,  bacteria  of,  202 

gonococci  in,  254 

methods  of  examination  of,  222 

nature  of,  200 

origin  of,  213 

pneumococci  in,  234 

typhoid  bacillus  in,  364 


Symptoms  caused  by  bacteria,  186 

Syphilis,  bacillus  of,  503 
lesions  in,  505 
serum  diagnosis,  131,  510 
spirochate  pallida  in,  503 
transmission  to  animals,  508 

Syringes  for  inoculation,  141,  142 

Tabes  mesenterica,  283 

Tarozzi's     method     of     anaerobic 

cultures,  68 
Taurocholate  media,  50 
Tertian  fever,  594,  595 
Test-tubes  for  cultures,  52 
Tetanolysin,  424 
Tetanospasmin,  424 
Tetanus,  415 

anti-serum  of,  429,  523  et  scq. 
intravenous  injection  of,  431 

cerebral,  428 

dolorosus,  428 

immunity  against,  429 

methods  of  examination  in,  432 

treatment  of,  430,  547 
Tetanus  bacillus,  416 

inoculation  with,  422 

isolation  of,  417 

spores  of,  417 

toxins  of,  191,  423 
Tetrads,  12 
Texas  fever,  638 
Theory  of  exhaustion,  548 

of  phagocytosis,  552 

of  retention,  548 

humoral,  548 
Thermophilic  bacteria,  19 
Thermostable  opsonins,  540 
Thionin-blue,  101,  105 
Thiothrix,  16 
Three-day  fever,  646 
Tick  fever,  African,  494 
Timothy-grass  bacillus,  278 
Tissues,  action  of  bacteria  on,  181 

fixation  of,  96 
Tizzoni  and    Cattani  on    tetanus, 

429 

Toxalbumins,  187 
Toxic  action,  theory  of,  198 
Toxicity,  estimation  of,  523 
Toxin  -  antitoxin   combination,    re- 
solution of,  527,  529 
Toxins,    concentrated,    method    of 
obtaining,  194 

constitution  of,  549 


INDEX 


687 


Toxins,  early  work  on,  187 

effects  of,  181 

immunisation  by,  518 

intra-  and  extra-cellular,  188 

nature  of,  193 

non-proteid,  194 

of  anthrax,    cholera,    etc.     (sec 
Special  Diseases) 

production,  179 

susceptibility  to,  549 

vegetable,  196 
Toxoids,  198,  527 
Trachoma,  bacteria  in,  219,  471 
Trichophyta,   media    for   growing, 

52 

Trophozoitea  (malaria),  587 
Tropical  ulcer,  636 
Trypanosoma  cnizi,  629 

gambiense,  623 

L-uisi,  617,  620 

noctuae,  614 

of  sleeping  sickness,  621 

ugandense,  610,  624 

ngandense,  relation  to  Tr.  Gam- 

biense,  628 

Trvpanosomata     associated      with 
various  diseases,  610 

biology  of,  610,  613 

culture  of,  43,  45,  612 

morphology  of,  610 

sexual  cycle  in,  614 
Ti  ypmosoniiasis,  610 
Tse-tse  fly  disease,  618 
Tubercle  bacillus,  262 

action  of  dead,  281 

avian,  276 

cultivation  of,  265 

distribution  of,  270 

immunity  against,  290 

inoculation  with,  273 

microscopic  methods.  295 

powers  of  resistance  of,  268 

in  sputum,  etc.,  281,  295 

toxins  of,  284 

<pecitii-  reactions,  _:>  l 

stains  for,  108,  264 

u'iant:  cells,  268 

met  hod  of  examination  of,   295 
Tubercles,  structure  of,  268 
Tubercular  leprosy,  298 
Tuberculin,  284,  288 

"  Bazillenemulsion,"  288 
Tuberculin,  "0"  and  "R,"  288 

therapeutic  application  of,  290 


Tuberculin  reactions,  284  ct  s>-q. 
Tuberculosis,  260 

in  animals,  261 

avian,  276 

bovine.  274 
its  relation  to  human,  2/4 

diagnosis  by  tuberculin,  287 

in  tish,  277 

immune-bodies    and    precipitins 
in,  288 

immunity    phenomena    in,    284, 
288 

modes  of  infection,  282 

precautions  in  diagnosis  of,  281 
Tubes,  cultures  in,  52 
Typhoid  bacillus,  356 

biological  reactions,  361 

comparison  with  b.  coli,  356 

culture  methods,  47,  49 

distribution  of,  368 

epidemiology  of,  369 

examination  for,  377 

immunity  against,  366 

inoculation  with,  365 

isolation    from    water    supplies, 
378 

occurrence     of     gallstones     in, 
364 

serum  diagnosis,  371 

suppuration  in,  364 

toxins  of,  366 

vaccination  against,  375,  377 
Typhoid  carriers,  369 
Typhoid  fever,  362 

pathological  changes  in,  362 
Typhus  fever,  648 

Ulcerative  endocarditis,  216 

experimental,  217 

gonococci  in,  256 
Unit  of  immunity,  524 
Urine,  examination  of,  74 

staining  of  bacteria  in,  94 

tubercle  bacilli  in,  272,  295 

typhoid  bacilli  in,  378 
1  "-< -hinsky's  medium  for  diphtheria 
bacilli,  407 

Vaccination  against  smallpox,  ^\\ 

nature  of,  571 
against  hydrophobia,  579 
against  typhoid,  375 
for  infection  by  pyogenic  bacteria, 
222 


688 


INDEX 


Vaccines,  preparation  of,  133 
Variola,  567  ct  seq. 
Vegetable  poisons,  196 
Venins,  197 
Vibrio  (see  also  Spirillum),  15 

berolinensis,  462 

of  cholera,  447 

Danubicus,  462 

Deneke's,  465 

Finkler  and  Prior's,  464 

Gindha,  463 

Ivanoff,  462 

Massowah,  454,  462 

Metchnikovi,  464 

Nordhafen,  464 

of  Pestana  and  Bettencourt,  463 

Romanus,  463 
Vibrion  septique,  433 
Vincent's  bacillus,  444 
Virulence,  attenuation  of,  515 

exaltation  of,  517 

of  bacteria,  176 
Voges    and    Proskauer's    reaction, 

353 

Volpino  on  smallpox,  571 
Von  Pirquet's  test,  285 

Wassermann   reaction   in  syphilis, 
131,  510 

in  general  paralysis,  510 

in  leprosy,  304 
Water,  bacteria  in,  156 

contamination     of,    by    sewage, 
169 

examination  of,  156 

supplies,  typhoid  bacilli  in,  378 
Weichselbaum  on  pneumonia,  226 
Weigert's  method  of  dehydration, 
100 

modification  of  Gram's  method, 

106 
Whooping-cough,  bacteria  in,  472 

culture  methods,  43,  44,  473 

inoculation  experiments,  474 

methods  of  examination,  475 

pathogenic  effects,  474 

serum  reaction,  474 
WTidal  on  serum  diagnosis,  541 


Widal's     reaction,     synonym     for 
agglutination  of  b.  typhosus, 
q.v.,  118,  382 
Williams   and    Lowden    on    Negri 

bodies,  576 
Winogradski,  24 
Winslow  and  Rogers  on  coccacea?, 

140 

Winter-spring  fevers,  595 
Wolff  and  Israel's  streptothrix,  326 
Woodhead  on  tuberculosis,  283 
Woody  tongue,  323 
Woolsorter's  disease,  343 
Wright's,      A.      E.,      bactericidal 

method,  127 
calibrated  pipette,  117 
diluting  pipette,  71 
method  of  counting  dead  bacteria, 

133 

opsonie  technique,  122 
vaccination  against  tuberculosis. 

291 
vaccination    treatment    of    pyo- 

genic  infections,  222 
Wright,      J.     H.,     on     anaerobic 

streptothrices,  324 
on  Leishmania  tropica,  63 
Romanowsky  stain,  113 

Xerosis  bacillus,  411 
Xylol,  100 

Yaws,  spirochsetes  in,  511 
Yellow  fever,  639 

bacteria  in,  640 

etiology  of,  640 

mosquitoes  in  relation  to,  641 
Yersin   (see  also  Roux)  on  plague, 

475,  486 
Yersin 's  anti-plague  serum,  487 

Ziehl-Neelsen  stain,  108 

Fraenkel's  modification,  108 
Ziemanni,  spirillum,  616 
Zone  phenomena  in  agglutination, 

543,  545 
Zoogloea,  3 
Zygote  (malaria),  592 


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